Method for Improving Quality of Aquaculture Pond Water Using a Nutrient Germinant Composition and Spore Incubation Method

ABSTRACT

A method for improving the quality of pond water used in aquaculture applications by adding to the pond water active bacteria that are preferably germinated from spores on site using a nutrient-germinant composition and an incubation method for increased spore germination efficiency, in combination with a nitrification enhancement agent such as calcium carbonate or calcified seaweed, and an optional reaction surface area modifier such as calcified seaweed or plastic or metal particles or fragments. The nutrient-germinant composition comprises L-amino acids, D-glucose and/or D-fructose, a phosphate buffer, an industrial preservative, and may include bacteria spores (preferably of one or more Bacillus species) or they may be separately combined for germination. The incubation method comprises heating a nutrient germinant composition and bacteria spores, to a temperature range of 35° C. to 60° C. for around 2 to 60 minutes to produce an incubated bacteria solution that is discharged to the aquaculture application.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.15/907,682 filed on Feb. 28, 2018, which is a continuation-in-part of(1) U.S. application Ser. No. 14/720,088 filed on May 22, 2015, now U.S.Pat. No. 9,908,799 issued on Mar. 6, 2018, which claims the benefit ofU.S. Provisional Application Ser. No. 62/002,476 filed on May 23, 2014and (2) U.S. application Ser. No. 15/479,773 filed on Apr. 5, 2017, nowU.S. Pat. No. 10,610,552 issued on Apr. 7, 2020, which claims thebenefit of U.S. provisional patent application No. 62/318,587 filed Apr.5, 2016.

BACKGROUND OF THE INVENTION 1. Field of the Invention

This invention relates to the treatment of aquaculture pond water withbacteria germinated in a nutrient germinant composition and using apoint-of-use spore incubation method to reduce organic waste, ammonia,and disease pressure in a water livestock application and to provideprobiotics to aquaculture species.

2. Description of Related Art

Aquaculture refers to the raising of aquatic species that are used as ahuman or animal food source. The technique applies some types of controlto the natural environment of the raised species to improve overallharvests. This can include the artificial hatching of species toincrease the commercial harvest of animals in the wild, hatching andraising of the species in enclosed ponds, and the hatching and raisingof species in tidally drained enclosed areas adjacent to the shoreline.Problems associated with this process include: pollution that isdischarged from the raising facility and will deteriorate the waterquality around; loss of product due to deteriorated water quality in theraising facility; and increased disease pressures associated withpathogenic microorganisms in the raising facility. Such problems may beidentified through testing or monitoring a variety of parameters,including pH, conductivity, ammonia, nitrate, phosphate and alkalinity.Conductivity is an indicator of salt content, amounts greater than 1200ppm is no longer considered fresh water; an ideal amount is 700 ppm andrange of 300-1200 ppm. Ammonia levels measure the amount of availableoxygen for fish. High levels of ammonia block oxygen transfer in fishfrom gills to the blood; however it is also a product of their metabolicwaste. While ammonia from fish waste is often not concentrated enough tobe toxic itself, fish farmers must closely monitor ammonia levels due tothe high concentration of fish per pond. Oxygen is consumed bynitrifying bacteria in the pond which break down the toxic ammonia to anon-toxic form; however, this massive use of oxygen reduces the oxygenavailable for uptake by fish. Ammonia levels>1 ppm are considered toxicfor fish life. Additionally, nitrate levels are examined to determinethe amount of plant fertilizer in the water. Nitrate is highly leachablefrom the surrounding soil and can be harmful to small children andpregnant women. Nitrate becomes nitrite in the GI tract and interactswith the blood's ability to carry oxygen. Max contamination level fornitrate is 10 ppm. Alkalinity is the measure of a pond's or lake'sability to neutralize acid without a change in pH. Alkalinity willdecrease over time due to bacteria; however an ideal level is 100 ppmwith acceptable range of 50-200 ppm. Phosphate found in ponds and lakesis largely from human and animal waste. Fertilizer run-off is a majorsource of phosphate found in golf course and decorative ponds. Elevatedlevels cause an increased rate of eutrophication which in turn increasessludge production. Moderate levels of phosphate can stimulate plantgrowth causing an increase in algae production; levels of >0.1 ppm is anindication of accelerated plant growth and is considered outsideacceptable levels.

Current technologies to address these problems include bioremediation,antibiotics, and chemical additives. Typical bioremediation technologiesinclude the application of supplemental bacteria to the water to enhancethe microbiological activities to improve the water quality. It is alsoknown to use nitrifiers to enhance the nitrification process to convertthe toxic ammonia into non-toxic nitrate. Chemical additives are addedto improve the water quality and aid the microbiological activities byproviding extra nutrients and alkalinity. Antibiotics are added toinhibit the growth of the pathogenic microorganisms. Problems associatedwith the current technologies include high cost and poor water qualityimprovement performance with the inactive supplemental bacteria, lownitrification activities due to the existence of organic waste and lackof nitrifier growing sites, and bioaccumulation of antibiotics in theraised aquatic species.

According to preferred methods disclosed in U.S. application Ser. No.14/720,088, active bacteria may be generated on-site using abiogenerator to grow the bacteria to a useful population from a solidbacteria starter material. The active bacteria may then be dischargedinto an aquaculture application from one or more biogenerators. Suchbiogenerators and their methods of use are disclosed, for example, inU.S. Pat. Nos. 6,335,191; 7,081,361; 7,635,587; 8,093,040; and8,551,762, the contents of which are incorporated by reference into thisdisclosure. However, it would be useful to have an alternate method ofgenerating active bacteria from spores at the point of use in anaquaculture application.

Spore germination is a multistep, causative process wherein sporeseffectively wake-up or are revived from a dormant state to a vegetativegrowth state. The first step is one by which spores are activated andare induced to germinate, typically by an environmental signal called agerminant. This signal can be a nutrient such as an L-amino acid.Nutrient germinants bind to receptors in the inner-membrane of the sporeto initiate germination. Additionally, sugars have been shown toincrease the binding affinity of L-amino acids for their cognatereceptors.

The germinant signal then initiates a cascade that causes the release ofDipicolinic Acid (DPA), which is stored in a 1:1 ratio with Ca²⁺ (CaDPA)in the core of the spore. The release of CaDPA is a fast process and istypically >90% complete in 2 min. CaDPA release represents a point of noreturn for spores in which they are committed to the germinationprocess. Those knowledgeable in the art refer to this step as the“commitment” step.

After CaDPA release, the spore is partially hydrated and the core pHrises to approx. 8.0. The core of the spore then expands and the cortex(composed mostly of peptidoglycan) is degraded by core lytic enzymes.The spore absorbs water and consequently loses its refractivity. Thisloss of refractivity towards the end of the germination process allowsspore germination to be monitored via phase-contrast microscopy.

The second phase of germination is an outgrowth step in which thespore's metabolic, biosynthetic, and DNA replication/repair pathwaysinitiate. The outgrowth period has several phases. The first is known asa ripening period in which no morphological changes (such as cellgrowth) occur, but the spore's molecular machinery (e.g. transcriptionfactors, translation machinery, biosynthesis machinery, etc.) isactivated. This period can vary in length based on the initial resourcesthat are packaged with the spore during the process of sporulation. Forinstance, the preferred carbon source of several Bacillus species(including subtilis) is malate and Bacillus spores typically contain alarge pool of malate that is used during the revival process.Interestingly, deletion mutants that cannot utilize the malate pooldisplay an extended ripening period compared to wild-type sporesindicating that the spore malate pool is sufficient to energize theinitial outgrowth process. Additionally, spores store small,acid-soluble proteins that are degraded within the first several minutesof revival that serve as an immediate source of amino acids for proteinsynthesis. After the outgrowth step, spore revival is complete and cellsare considered to be vegetatively growing.

It is known that spores can be induced to germinate via heat-activation.Spores of various Bacillus species have been heat-activated atstrain-specific temperatures. For example, B. subtilis spores have beenheat-activated at 75° C. for 30 minutes while B. licheniformis sporeshave been heat-activated at 65° C. for 20 minutes. The heat-activationhas been shown to cause a transient, reversible unfolding of spore coatproteins. Heat-activated spores can then be germinated for additionaltime in germination buffers containing nutrient germinants, such asL-alanine. If no nutrient germinant is present, however, spores willreturn to their pre-heated, non-germinated state.

It is also known that germination can occur at ambient temperatures(near typical room temperature) without heat-activation and with agermination buffer containing nutrients, but the process usually takeslonger than with heat-activation. For example, B. licheniformis and B.subtilis spores will germinate at 35° C. or 37° C., respectively, but ittakes a longer period of time (e.g. 2 hours) in a germination buffercontaining nutrient germinants. Additionally, non-heat-activated sporesof B. subtilis have been known to have been germinated in non-nutrientgerminant conditions (e.g. CaCl₂+Na₂DPA) for an extended period of time.

It is also known to combine the use of heat activation and a nutrientgerminant to germinate spores in a two-step process in laboratorysettings. The spores are first heat activated by incubating for a periodof time (e.g. 30 minutes) at a temperature in the range of 65-75° C.(this specific temperature is species dependent). Then, the spores aretransferred into a buffer solution that contains a nutrient germinant,such as L-alanine. It is also known to grow bacteria in a growth chamberlocated near a use site by feeding pelletized nutrient material(containing sugar, yeast extract, and other nutrients that are notdirect spore germinants), bacteria starter, and water into a growthchamber at a controlled temperature range of 16-40° C., and morepreferably between 29-32° C., for a growth period of around 24 hours asdisclosed in U.S. Pat. No. 7,081,361.

There is a need for a rapid spore incubation and activation method thatwill allow generation of active bacteria, such as Bacillus species, in asingle step at a point-of-use location where the bacteria will bedischarged into an aquaculture application. Accordingly, this inventiondescribes a simple method for spore germination using a nutrientgerminant concentrate combined with a spore composition, or using anutrient spore composition, simultaneously with heat incubation in asingle step for use in aquaculture applications.

SUMMARY OF THE INVENTION

The method of the invention provides a cost-effective approach todelivering active bacteria to pond water (or a growing pond) in anaquaculture facility to degrade the organic waste and inhibit the growthof pathogenic microorganisms without bioaccumulation. The method of theinvention reduces disease pressure in the water livestock, resulting inimproved harvests of the species raised in the aquaculture operation.The addition of a nitrification enhancement agent as disclosed here aspart of the method provides a steady alkalinity source and extra growingsites for the nitrifier to promote the nitrification activity andammonia reduction.

The method of the invention desirably includes the delivery of activebacteria, optionally including a probiotic bacteria, most preferablygenerated from an on-site incubator using a liquid nutrient germinantconcentrate and bacteria in spore form, into an aquaculture application.A nutrient-germinant composition according to one preferred embodimentof the invention comprises one or a combination of many L-amino acids,optionally D-glucose (which increases the binding affinity of L-aminoacids for their cognate receptors in the spore coat), and a neutralbuffer such as a phosphate buffer, and an industrial preservative, suchas the commercially available Kathon/Lingaurd CG (which has activeingredients comprising methyl chloro isothiazolinone and methylisothiazolinone). A nutrient-germinant composition according to anotherpreferred embodiment of the invention comprises one or a combination oftwo or more L-amino acids, optionally D-glucose (which increases thebinding affinity of L-amino acids for their cognate receptors in thespore coat), HEPES sodium salt (a biological buffer to provide theproper pH for spore germination), and an industrial preservative, suchas a combination of propylparaben and methylparaben or other U.S.federal GRAS (Generally Regarded As Safe) preservatives. According toanother preferred embodiment, the spore composition also comprises asource of potassium ions, such as potassium chloride or monopotassiumphosphate or dipotassium phosphate. According to another preferredembodiment, the spore composition includes both D-glucose andD-fructose.

According to another preferred embodiment, a nutrient germinantcomposition also comprises spores of one or more bacteria species,preferably Bacillus species but other bacteria may also be used, andincludes a germination inhibitor, such as NaCl, industrialpreservatives, or D-alanine, in combination with any of the previouslydescribed spore composition ingredients. The germination inhibitorprevents the spores from germinating prematurely in thenutrient-germinant composition. The germination inhibitor may includechemicals that prevent spore germination such as NaCl, industrialpreservatives, or D-alanine.

Alternatively, bacterial spores may be separately provided and added toa nutrient-germinant composition according to the invention at thepoint-of-use and incubation. When separately added, it is preferred toprovide a stable spore suspension spore composition comprising one ormore bacteria species, preferably Bacillus species. According to onepreferred embodiment, a spore composition comprises bacteria spores,about 0.00005 to 3.0% by weight surfactant, about 0.002 to 5.0% byweight thickener, and optionally about 0.01 to 2.0% by weight ofacidifiers, acids, or salts of acids (including those used as apreservative or stabilizer), with the balance being water. According toanother preferred embodiment, a spore composition comprises bacterialspores, about 0.1 to 5.0% by weight thickener, about 0.05 to 0.5% byweight acids or salts of acids, optionally about 0.1-20% by weight wateractivity reducers, and optionally about 0.1% to 20% additional acidifier(acids or salts of acids), with the balance being water.

Most preferably, the bacterial spores in both preferred sporecomposition embodiments are in a dry, powder blend of 40-60% salt (tablesalt) and 60-40% bacteria spores (prior to adding to the sporecomposition) that combined make up about 0.1 to 10% by weight of thespore composition. The spore compositions preferably comprise around1.0×10⁸ to around 3.0×10⁸ cfu/ml of the spore composition (sporesuspension), which when diluted with drinking water (for animal wateringapplications) provide around 10⁴ to 10⁶ cfu/ml bacterial strains in thedrinking water. Most preferably, the thickener in both preferredembodiments is one that also acts as a prebiotic, such as xanthan gum,to provide additional benefits. Although other commercially availablespore products may be used, preferred spore compositions for use withthe invention are as disclosed in U.S. application Ser. No. 14/524,858filed on Oct. 27, 2014, which is incorporated herein by reference.

According to another preferred embodiment, a nutrient germinantcomposition according to the invention is in concentrated form and isdiluted to 0.01% to 10% strength in water or another diluent at thepoint-of-use. The use of a concentrated formula reduces shipping,storage, and packaging costs and makes dosing of the spore compositionat the point-of-use easier. Most preferably, the concentrated sporecomposition is in a liquid form, which is easier and faster to mix withdiluent at the point-of-use, but solid forms such as pellets or bricksor powder may also be used. The inclusion of a general, industrialpreservative in the spore composition aids in long-term storage and/orgermination inhibition, which is particularly useful when the sporecomposition is in the preferred concentrated form.

In another preferred embodiment, the present invention comprises amethod of germinating spores of Bacillus species using a nutrientgerminant composition combined with a spore composition or using anutrient spore composition at an elevated temperature; preferably in arange of 35-60° C., more preferably in the range of 38-50° C., and mostpreferably in the range of 41° C. to 44° C. for a period of time (anincubation period). The incubation period preferably ranges from 2-60minutes, or longer, depending on the application. Most preferably, anutrient-germinant composition or nutrient spore composition inconcentrated form according to preferred embodiments of the inventionare used in the incubation/germination methods of the invention, butother nutrient-germinant compositions and spore compositions may also beused. Preferably, the incubation method is carried out at or near thepoint-of-use—the aquaculture site or near the aquaculture site where thegerminated spores will be used or consumed and further comprisesdispensing the germinated spores to the point-of-use/consumption.Preferred methods according to the invention may be carried out in anyincubation device that has a reservoir capable of holding a volume ofspores (if separately added), liquid (typically water as a diluent),nutrient-germinant composition and that is capable of heating themixture during an incubation period. Most preferably, the methods arecarried out in a device that is also capable of mixing thoseingredients, automatically shutting-off heating at the end of theincubation period, and automatically dispensing an incubated bacteriasolution comprising the bacteria to an aquaculturepoint-of-use/consumption. Preferred methods may also be carried out as abatch process or as a continuous process. Although spore compositionsaccording to the invention are preferably used, any variety of sporeforms or products, such as dried powder form, a liquid suspension, or areconstituted aqueous mixture, may be used with the method of theinvention.

The preferred embodiments of the invention allow for rapid germinationof spores of Bacillus species at an aquaculture point-of-use. Theactive, vegetative bacteria solution discharged from the incubator canbe supplied directly to growing ponds or can be accumulated and dilutedwith pond water or another similarly suitable diluent, such as waterfrom a municipal water system, prior to discharging it into growingponds. Alternatively, the incubator may be configured to heat a nutrientgerminant composition and spores, or a nutrient spore composition, foran incubation period and temperature range that will produce bacteria ina metastable state, between dormant spores and vegetative bacteria. Themetastable bacteria solution is then discharged into the growing pondwhere the bacteria are able to become active, vegetative bacteria.Dilution may aid in delivery of the treatment solution flowing from theincubator to the growing pond, if the incubator is located some distancefrom the growing pond. The active bacteria will degrade the organicwaste and inhibit the growth of the pathogenic microorganisms in thewater in the aquaculture facilities, without requiring the addition of(or reducing the amount of) chemical treatments and antibiotics used inthe growing pond.

The invention also desirably includes contemporaneous application of atleast one nitrification enhancement agent to the growing ponds.Nitrification enhancement agents increase the activity of nitrifyingbacteria naturally found in the water to decrease the ammonia level.These nitrification agents comprise alkalinity enhancement agents thatincreasing the alkalinity of the water, which is necessary fornitrification (7 parts alkalinity to 1 part ammonia) and/or surface areamodifying agents to add surfaces for nitrifying bacteria to grow, sincenitrifying bacteria grow as biofilms and need surfaces to which they canattach. The alkalinity enhancement agents can include, for example,calcium carbonate, calcified seaweed or other similarly effectiveadditives. These agents can be added at a higher-than-dissolution amountto provide a continuing source of alkalinity as they slowly dissolve.Certain nitrification agents, such as calcified seaweed act as both analkalinity enhancement agent and a surface area modifying agent byproviding both alkalinity and high surface area, providing a supportsurface for biofilms of nitrifiers to grow. Calcified seaweed also actsas a source of micronutrients for the bacteria. Other nitrificationenhancement agents only act as surface area modifying agents, such asplastic or metal pieces, or other similarly effective materials toincrease the surface area over which favorable reactions andinteractions can occur. One or more agents that act only as surface areamodifiers (and not alkalinity enhancers) may also be added to thegrowing pond, either alone or preferably in combination with one or morealkalinity enhancement agents; however, an agent that acts only as asurface area enhancer would not degrade in the growing pond and wouldnot be added with each batch of bacteria solution. Such agents that actonly as surface area modifiers would preferably only be added to agrowing pond once. Agents that act as alkalinity enhancement agentswould be added to the growing pond contemporaneously with a batch ofbacteria on a periodic basis, such as seasonally (once per season oronce every summer, twice per year, etc.) or as needed. As used herein,the term “contemporaneous” is intended to mean “at or about” the timethat a batch of vegetative bacteria and other components or agents areadded to the growing pond or other growing medium in which aquaticspecies are grown at an aquaculture facility.

BRIEF DESCRIPTION OF THE DRAWINGS

The system and method of the invention are further described andexplained in relation to the following drawings

FIG. 1 is a flow diagram for an incubation system and method accordingto a preferred embodiment of the invention;

FIG. 2 is a flow diagram for an incubation system and method accordingto another preferred embodiment of the invention;

FIG. 3 is a flow diagram for an incubation system and method accordingto another preferred embodiment of the invention;

FIG. 4 is a graph of nitrate levels in a laboratory study;

FIG. 5 is a graph of ortho-phosphate levels in a laboratory study;

FIG. 6 is a graph of turbidity in a laboratory study

FIG. 7 shows photographs of bacteria slides using a spore compositionand method according to a preferred embodiment of the invention comparedto control slides;

FIG. 8 is a graph showing pO₂ test data to demonstrate germinationlevels using a spore composition and method according to a preferredembodiment of the invention compared to control tests;

FIG. 9 is a graph showing pO₂ test data to demonstrate germinationlevels using a spore composition and varied methods according topreferred embodiments of the invention compared to control tests; and

FIG. 10 shows an image of three aquariums, each under control (left),calcium carbonate only treatment (middle), or treatment with anactivated nutrient-spore formulation (right).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Aquaculture Treatment Methods

According to one preferred embodiment, active bacteria are generated onsite from a nutrient germinant composition combined with a sporecomposition or from a premixed nutrient spore composition, preferablyusing an incubator system and a preferred germination method asdescribed below, and the active bacteria are periodically fed into agrowing pond in an aquaculture application. One or more nitrificationenhancement agents are also contemporaneously added to the growing pondwith the active bacteria.

A satisfactory bacteria growing and delivery device for use in themethod of the invention will include an on-site incubator system, suchas an air incubator, a water incubator, or any other chamber or similardevice that provides even, constant heat at a given temperature range asneeded to germinate the spores for discharge into the aquacultureapplication. Referring to FIG. 1, preferably, the on-site incubatorsystem 10 contains one or more tanks or holding containers for holdingan initial volume of nutrient germinant composition 12 and an initialvolume of bacteria spore solution 14 (if the bacterial spores are notincluded in the nutrient germinant composition). These sporecompositions may also arrive at the site of use in containers that areconnectable in fluid communication with the incubator, in which caseseparate tanks or containers are not needed. A source of water 16 at ornear the aquaculture site is also optionally, but preferably,connectable in fluid communication with the incubator system. A well,source of municipal water supply, or the growing pond may provide water16 to the incubator 18. An incubator system 10 also preferably comprisesa chamber or container 18 configured to receive a portion of thenutrient germinant composition and spores and allow them to be heated togerminate the spores; a heater; valves, tubing, and pumps (as needed, ifgravity flow is not sufficient) to allow the nutrient germinantcomposition 12, bacteria spore solution 14, and optionally water 16 toflow from their storage containers/source into a heating chamber orcontainer 18 and to discharge an incubated bacteria (or activatedbacteria) solution 20 from the heating chamber or container 18 anddeliver it to the growing pond 22; an optional mixer within the heatingchamber or container 18, and a controller or timer to activate thevalves, pumps, optional mixer, and heater to control influx of thenutrient and spore compositions into the heating chamber, incubationtime and temperature, and discharge to the growing pond. Mostpreferably, the on-site incubator system 10 uses a nutrient germinatecomposition combined with a bacteria spore composition (as describedbelow) or uses a nutrient spore composition (a nutrient germinantcomposition pre-mixed with bacteria spores, also as described below)(either are also referred to herein as “starter material”), to generatean incubated bacteria solution 20 to be discharged into the growing pond22.

Alternatively, according to another preferred embodiment as shown inFIG. 2, incubator system 110 uses concentrated nutrient germinantcomposition 24 and concentrated spore composition 30, which are dilutedwith a diluent or water from container/source 26 to form a workingnutrient germinant composition 28 and a working spore composition 32, aportion of each being fed into incubator 18 to generate a batch ofactivated bacteria 20. Water from source 16 may also be used as a sourceof diluent in place of or in addition to source 26. Additionally, onlyone of the nutrient germinant composition 24 or spore composition 30 maybe in concentrated form and require dilution prior to feeding intoincubator 18. According to another preferred embodiment, when only oneis concentrated, the non-concentrated composition may be used as adiluent for the concentrated composition in addition to or in place ofwater/diluent from source 26 and/or source 16. According to anotherpreferred embodiment, a concentrated nutrient spore composition 34 isused with system 210 as shown in FIG. 3. The concentrated nutrient sporecomposition is diluted with water/diluent from source 26 and/or source16 to make a working nutrient spore composition 36 prior to feeding intoincubator 18 to generate a batch of activated bacteria 20.Alternatively, a nutrient spore composition may not be in concentratedform and not require any diluent prior to feeding into incubator 18(similar to direct feeding of nutrient germinant composition 12 in FIG.1), in which case water/diluent source 26 is not needed. Water fromsource 16 may still be optionally fed into incubator 18 as needed inthis alternate embodiment. With any of the incubator system embodiments,diluent may be fed into incubator 18 to dilute a concentratedcomposition within the incubator rather than prior to feeding theincubator. Any combination of elements from systems 10, 110, and 210 maybe used together, as will be understood by those of ordinary skill inthe art.

Preferably, bacteria spores are germinated in incubator 18 or othersuitable heating device according to preferred germination methodsdescribed herein. According to one preferred embodiment, a nutrientgerminant composition and spore composition (or nutrient sporecomposition) are heated in incubator 18 to a temperature in a range of35-55° C., more preferably in the range of 38-50° C., and mostpreferably in the range of 41° C. to 44° C. The incubation period canvary depending on the end-use application, but is preferably betweenaround 20 minutes to 60 minutes to generate active bacteria for anaquaculture application and most preferably around 2 minutes to 5minutes for a probiotic application to generate metastable statebacteria. To provide additional growth time for vegetative bacteria, theincubation period may be around 4 to 6 hours.

Depending on the desired use of the bacteria in the aquacultureapplication, such as use to treat the water or a probiotic for theaquatic species, different incubation periods may be used to provide anincubated bacteria solution that is primarily still spore form bacteria,primarily metastable state bacteria (in which the spores are neitherdormant nor in the vegetative growth phase, also referred to herein asan activated state), or primarily fully vegetative bacteria.Additionally, when fully vegetative bacteria are desired, the bacteriasolution may be held within the incubator 18 or another intermediatecontainer for a period of time after the incubation period to allow thebacteria multiply prior to discharging into the aquaculture application.Most preferably, the bacteria solution will be maintained at atemperature between 30 to 45° C., more preferably, the vegetativebacteria solution will be heated as necessary to maintain thetemperature of the solution in the range of 33 to 42° C., and mostpreferably in the range of 36° C. to 39° C. to facilitate growth duringthis post incubation growth period. When a probiotic application isdesired, the bacteria remain primarily in the spore state or metastablestate when discharged to the aquaculture application by using a shorterincubation period, which gives the bacteria a better chance of survivingthrough to the aquatic species' intestinal tract where they are mostbeneficial as probiotics. At the end of an incubation period, anincubated bacteria solution 20 is discharged to the growing pond. Anincubated bacteria solution 20 may comprise fully vegetative bacteria,metastable state bacteria, spores, or a combination thereof depending onthe species of bacteria used, incubation temperature, incubation time,and content of the nutrients used.

Each batch of incubated bacteria solution 20 comprises around1×10⁸-1×10¹⁰ cfu/mL of metastable state, vegetative bacteria species,and/or spores. Once discharged into growing pond 22, the amount ofbacteria in each batch is diluted based on the amount of water in thegrowing pond. Most preferably, sufficient quantities of bacteriasolution 20 are added to the growing pond 22 to provide an effectiveamount of activated bacteria based on the dilution in the growing pond.In this context, “effective amount” can refer to the amount of bacteriaand/or nutrient composition that can be effective to improve performanceof a plant or animal after administration. An improvement in performancecan be measured or evaluated by monitoring one or more characteristics,including but not limited to water quality: clarity of water, ammonialevels, nitrite levels, nitrate levels, disease incidence, mortality,harvest weight, meat quality, individual animal size, premium weights,antibiotic use, and additive use. “Effective amount” can also refer tothe amount that can reduce the amount of, competitively exclude, and/oreliminate one or more species of pathogenic bacteria (including, but notlimited to Escherichia coli and Salmonella) in the intestines of ananimal. “Effective amount” can also refer to the amount that can reduceNH₃ and/or H₂S levels, such as that which can be excreted by an animalinto its environment.

According to one preferred embodiment for use in shrimp aquacultureapplications, the effective amount of the bacteria in the growing pondcan be about 1 to about 9×10² CFU/mL. According to another preferredembodiment, the effective amount for shrimp aquaculture applications isabout 1 to about 9×10² to about 10⁸ CFU/mL. According to anotherpreferred embodiment, the effective amount of the incubated bacteria inthe growing pond can range from about 0.001% to about 2% v/v of thetotal amount of water in the growing pond and any range or valuetherein. As another example, around 500 mL of incubated bacteriasolution comprising around 1×10⁹-1×10¹⁰ cfu/mL of bacteria species dosedto a growing pond four times per day will be sufficient treat a growingpond containing 100,000 gallons of water. Other volumes of bacteriasolution and dosing intervals may be used to treat growing ponds,depending on the size of the pond, based on pond conditions, aquaticspecies, temperature of the pond, and other factors to achieve a desiredeffective amount of bacteria in the pond as will be understood by thoseof ordinary skill in the art.

Multiple incubator systems 10, 110, or 210 may be provided to providelarger quantities of incubated bacteria solution to the growing pond toachieve the desired effective amount being added to the pond, to providedifferent species of bacteria to the growing pond or at different timesor rates, and/or to space out the discharge of incubated bacteriasolution around the perimeter of the growing pond to aid in dispersingthe bacteria through the pond. A pump or other mixing device may also beadded to the growing pond (if not already in place) to aid in dispersingthe incubated bacteria solution (and nitrification enhancers or surfacearea enhancers) throughout the growing pond.

The on-site incubator is preferably configured to incubate multiplebatches of incubated bacteria solution from a container of a nutrientgerminant composition/spore composition or nutrient spore composition,so that multiple batches of bacteria can be discharged at periodicintervals over a prolonged period of time before the starter materialneeds to be replenished. For example, a container of nutrient germinantcomposition 12 may initially hold 0.3 to 3 liters of nutrient germinantcomposition that may be fed to an incubator in incremental amounts ofaround 10 to 100 mL every 1 to 24 hours. A container of concentratednutrient germinant solution 24 may initially hold 0.2 to 1 liters ofsolution, be diluted with water/diluent from source 26 or 16 to a ratioof around 1:50 to around 1:10 concentrate to water/diluent to feed anincubator in incremental amounts of around 0.1 to200 mL of workingnutrient germinant solution 28 every 1 to 24 hours. A container ofbacteria spore solution 14 to be fed separately with a nutrientgerminant composition may initially hold 0.6 to 6 liters of solutionthat may be fed to an incubator in incremental amounts of around 20 to200 mL every 1 to 24 hours. A container of concentrated bacteria sporesolution 30 may initially hold 0.15 to 6 liters of solution, be dilutedwith water/diluent from source 26 or 16 to a ratio of around 1:10 toaround 1:3 concentrate to water/diluent to feed an incubator inincremental amounts of around 5 to 200 mL of working spore solution 32every 1 to 24 hours. A container of nutrient spore composition mayinitially hold 3 to 6 liters of nutrient germinant composition that maybe fed to an incubator in incremental amounts of around 100 to 200 mLevery 1 to 24 hours. A container of concentrated nutrient spore solution34 may initially hold 0.3 to 3 liters of solution, be diluted withwater/diluent from source 26 or 16 to a ratio of around 1:10 to around1:50 concentrate to water/diluent to feed an incubator in incrementalamounts of around 10 to 100 mL of working nutrient spore solution 36every 1 to 24 hours. Each batch of nutrient germinantcomposition/bacteria spores or nutrient spore composition is thenincubated in the incubator, as discussed herein, to form an incubatedbacteria solution that is discharged to an aquaculture application.

An incubated bacteria solution 20 is preferably discharged from one ormore incubators 18 to the growing pond 22 once every 4 to 6 hours overthe course of a treatment cycle. Other dosing intervals may be useddepending on the size of the pond, conditions of the pond/aquaticspecies, and type of application. The time between doses may be variedas desired by varying the timing of addition of ingredients to theincubator and/or incubation time. An incubated bacterial solution may bedischarged more frequently on a larger pond (e.g. 20 million gallons).For an aquaculture water treatment application, it is preferred todischarge an incubated solution having vegetative bacteria. To achievevegetative bacteria, it is preferred to incubate for at least 4 to 6hours before discharging to the growing pond, although longer incubationtimes to allow more time for the bacteria to multiply may also be used.For a probiotic application for aquatic species in an aquacultureapplication, it is preferred to incubate for around 2 to 5 minutes. Inthat application, an incubated bacteria solution 20 may be dischargedmultiple times a day, even as frequently as every 4 to 6 minutes, ifneeded for a large pond. The volume of nutrient germinantcomposition/spore composition or nutrient spore composition feeding theincubator is periodically replaced or replenished as needed. A treatmentcycle is preferably continuous with the incubator running throughout theyear (other than periodic shut-downs for maintenance or replenishment ofthe nutrient germinant composition).

Various Bacillus species, as described below, are preferably used withaquaculture treatment methods according to the invention, but otherbacteria may also be used. For example, the genera of bacteria suitablefor use in the method of the invention are believed to include any oneor more species in the genera Bacillus, Bacteriodes, Bifidobacterium,Lueconostoc, Pediococcus, Enterococcus, Lactobacillus, Megasphaera,Pseudomonas and Propionibacterium. Probiotic bacteria that may begenerated on-site include any one or more of the following: Bacillusamylophilus, Bacillus licheniformis, Bacillus pumilus, Bacillussubtilis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillusmegaterium, Bacteriodes ruminocola, Bacteriodes ruminocola, Bacterioidessuis, Bifidobacterium adolescentis, Bifidobacterium animalis,Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacteriumlongum, Bifidobacterium thermophilum, Enterococcus cremoris,Enterococcus diacetylactis, Enterococcus faecium, Enterococcusintermedius, Enterococcus lactis, Enterococcus thermophiles,Lactobacillus brevis, Lactobacillus buchneri, Lactobacillus bulgaricus,Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus curvatus,Lactobacillus delbruekii, Lactobacillus farciminis, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillusplantarum, Lactobacillus reuteri, Leuconostoc mesenteroides, Megasphaeraelsdennii, Pediococcus acidilacticii, Pediococcus cerevisiae,Pediococcus pentosaceus, Propionibacterium acidipropionici,Propionibacterium freudenreichii, and Propionibacterium shermanii.

With at least one dose (or batch) of incubated bacteria solutiondischarged to the growing pond, one or more nitrification enhancementagents are preferably added contemporaneously. Alkalinity enhancingagents, including calcium carbonate or calcified seaweed, may be addedperiodically, such as seasonally or as needed to reduce phosphates, andnot with each dose of bacteria. The agents can be added at ahigher-than-dissolution amount to provide a continuing source ofalkalinity as they slowly dissolve. Slowly dissolving alkalinityenhancing agents, such as calcified seaweed, also act as a surface areamodifier, providing a support surface for biofilms of nitrifyingbacteria to grow and they also aid in nutrient delivery. Additionally,agents that act only as surface area modifiers (such as pieces of metalor plastic) may be added to the growing pond as needed to reducenitrogen or phosphorous, along with a batch or dose of incubatedbacteria solution and one or more alkalinity enhancing agents, but thereare preferably added only once and not with each dose of incubatedbacteria solution. These surface enhancement agents similarly provide asupport surface for biofilms of the added bacteria to grow, which aidsin faster development of the beneficial bacteria. Most preferably,around 100 pounds of such nitrification enhancement agents are added per7.5 million gallons of growing pond, and this amount may be scaled forother growing pond volumes. Preferred dispersal methods for thenitrification enhancement agents can include the use of automateddevices or manual application to the water in the growing ponds.Automated or manually operated devices useful for broadcasting orotherwise dispersing at least one nitrification enhancement agent in theform of prills, pellets or granules are commercially available and arewell known to those of skill in the art. Additionally, thesenitrification enhancing agents may be dispersed through a pond using theself-dispersing additive system and method, which employs effervescentmaterials along with the treatment agent in water soluble packaging,described in U.S. patent application Ser. No. 14/689,790 filed on Apr.17, 2015, which is incorporated herein by reference.

Suitable applications for the method of the invention include, forexample and without limitation various types of aquaculture applicationsuch as hatcheries, ponds, and tidal flow aquaculture. The combined useof germinated or vegetative bacteria, preferably grown on-site, and atleast one nitrification enhancement aid such as calcium carbonate,calcified seaweed or another material that is similarly effective forcost-effective treatment of water used in aquaculture applications toaddress organic waste, ammonia, and pathogenic microorganism as well asgeneral water quality issues. The effectiveness of the subject methodfor achieving these objectives is believed to be further enhanced by theaddition of calcified seaweed, or other plastic or metal pieces,particles or fragments that increase the available surface area uponwhich interactions or reactions can occur.

A laboratory study was conducted to evaluate the benefits of addingbeneficial bacteria and nitrification enhancement agents to pond water.One goal of the study was to evaluate the efficacy of added bacteria(pond blend commercially available from EcoBionics) as inhibitory orherbicidal against algae production. This study employed the use of six2 L beakers, each filled with 1.5 L of source water taken from anestablished fish tank with algae present. Each beaker also contained onegold fish from a source tank, air stone, light source that alternated 12hours on, then 12 hours off, and watch glass cover to reduce loss toevaporation. A pond blend bacteria solution was generated in a BIO-Amp™biogenerator using 37 g of Pond Plus pellets, rather than using anincubator and nutrient germinant composition according to preferredembodiments of the invention described below. After a 24 hour growthcycle in the biogenerator, an aliquot of bacteria solution was obtainedand diluted to maintain a ratio of 3 L of pond blend: 579024 gallonspond water, however, a preferred ratio to be employed in the field is 3L of pond blend:100,000 gallons of pond water. Based on this ratio,around 0.4 μL of pond blend bacteria solution was added to specific ofthe beakers having 1.5 L of fish tank water. Calcified seaweed, wasadded to specific beakers according to manufacturer's instructions basedon rates for clarification; this equated to 0.045 g of calcified seaweedper 1.5 L of water. An equal amount of calcium carbonate was added tosome of the beakers. The additives in each beaker were as follows:

TABLE 1 Beaker 1 0.4 μL of pond blend and 1.5 L of source water onlyBeaker 2 0.045 g of calcified seaweed and 1.5 L of source water onlyBeaker 3 0.4 μL of pond blend, 0.045 g of calcified seaweed, and 1.5 Lof source water Beaker 4 0.045 g of calcium carbonate and 1.5 L ofsource water only Beaker 5 0.4 μL of pond blend, 0.045 g of calciumcarbonate, and 1.5 L of source water Beaker 6 served as the negativecontrol and contained only 1.5 L of source water

Each test beaker was treated according to one preferred dosing schedulethat would be utilized in the field. Beakers 1, 3 and 5 with pond blendwould be treated (dosed) once per week with an additional 0.4 μL of pondblend. Depending on growing pond conditions, other dosing schedules maybe used in the field. Calcified seaweed and calcium carbonate were addedonly once at the beginning of this study, however, additional dosing maybe used in the field.

One pre-treatment sample (before the addition of pond blend bacteriasolution, calcified seaweed or calcium carbonate) was taken from eachbeaker and analyzed to obtain a baseline for comparison to the posttreatment results. Chemical analysis was performed once per week using200-300 mL samples from each beaker. These weekly measurements includedanalysis of pH, conductivity, nitrate, ortho phosphate, total alkalinityand ammonia levels. Once per month turbidity was examined andphotographs were taken to assess changes in algal growth and overallclarity. Treatment and analysis of the beakers was continued for a totalof three months; again to mirror the length of the field study.

Data analysis was performed using Excel 2003, using a two sampledtwo-tailed t-test comparing pre-treatment vs. post-treatment numbers at95% confidence level. The two sample two-tailed t-test tested the nullhypothesis of no difference in the means of pre and post-treatment withan alternative hypothesis of there is a difference in the means.

H_(O)=μ pre-treatment=μ post-treatment

H_(A)=μ pre-treatment≠μ post-treatment

Baseline readings indicated elevated phosphate levels in all beakers,over 40 times the level indicative of accelerated algal growth. Allother measurements were within acceptable ranges.

TABLE 2 Results from Two Sample T-Test for Nitrate and Phosphate % %Nitrate Reduction Phosphate Reduction Beaker p-value Nitrate p-valuephosphate 1 (pond blend only) 0.88 20 0.47 38 2 (Calcified 0.01* 790.02* 73 Seaweed only) 3 (pond blend + 0.04* 76 0.07 66 calcifiedseaweed) 4 (Calcium 0.03* 77 0.05 66 carbonate only) 5 (pond lend + 0.2452 0.02* 72 calcium carbonate) 6 (control) 0.90 25 increase 0.68 19*Indicates a significant result

The test beaker containing only the bacterial blend pellets showed nostatistically significant change over the three month study period.Phosphate levels dropped after two weeks but within one month returnedto pre-treatment levels. Nitrate levels mirrored those of phosphate. Ofall the test beakers, only beaker 1 had nitrate and phosphate levelsrise similar to the negative control. This indicates a minimal effect onmajor chemical indicators of pond health when using bacteria alone.

The two beakers containing calcified seaweed showed statisticallysignificant changes in pre vs. post-treatment means. There was asignificant drop in nitrate levels of 79% and 76% with p-values 0.01 and0.04 in beaker 2 and 3 respectively. Phosphate levels also droppedsignificantly in beaker 2 by 74% with a p-value of 0.02. The beakercontaining calcified seaweed and pellets showed a phosphate reduction of66%, however, this value was not significant (see Table 2). It isimportant to note that this lack of statistical significance may besubject to this study's low sample size limitation. This study did nottest for changes in pathogenic bacteria in the samples, but the additionof the pond blend bacteria solution would be expected to reduce thosenumbers through competition. Additionally, it is believed that theaddition of a bacteria solution from an incubator using a nutrientgerminant composition into to an actual growing pond according to apreferred embodiment of the invention would achieve better results thanin the laboratory study because the bacteria in the bacteria solutioncan act synergistically with the nitrifying bacteria already present inthe growing pond and the added bacteria in the bacteria solution can aidin consuming waste in the water to reduce ammonia levels. Similar to thecalcified seaweed beakers, the two beakers that contained calciumcarbonate showed a significant difference in pre versus post treatmentmeans. In beaker 4, the nitrate levels dropped 77% with a p-value of0.03. While, beaker 5 which contained calcium carbonate and bacterialpellets showed a significant decrease in phosphate levels of 72%,p-value 0.02 (see Table 2). Calcium carbonate may be a suitablesubstitute for calcified seaweed in aquaculture treatment. Beakers withcalcified seaweed or calcium carbonate out performed those without.Beaker 5, which contained bacterial blend pellets and calcium carbonate,had a significantly lower post-treatment mean of phosphate levels andthe least effect on pH. However, beaker 2 and 5 had statisticallysignificant drops in phosphate levels.

Turbidity examined throughout this study showed a continual decrease inall the test beakers. When comparing pre-treatment pictures to post,there is an increase in the presence of algae in all beakers, however,by the second month there was evidence of algal death in two beakers.Beaker 4 containing calcium carbonate and Beaker 6 (control) bothappeared yellow in color indicating a dying algal system. Algal death isa common problem experienced after an initial algal bloom, as oxygen inthe water is depleted; despite the presence of the air stone. As thealgae died, there was a marked increase in nitrate levels. This wasevident by the increase in nitrate levels above pre-treatment in thismonth, increasing 14% and 38% in beaker 4 and 6 respectively. By thefinal month, nitrate levels in Beaker 4 recovered and decreased thoughthe system still had a dark green, yellow color. Nitrate continued toincrease to 6× the contamination level in Beaker 6. FIGS. 4-6 are graphsshowing the results of this laboratory study.

A field study focusing on improving general pond health and claritywhile reducing sludge was also conducted on various ponds. Although thisstudy was not aquaculture specific (as the ponds in the study wereornamental or recreational and not for raising and harvesting aquaticspecies) and used a biogenerator rather than an incubator and a nutrientgerminant composition according to a preferred embodiment of theinvention, it provides some useful information on the addition ofbacteria and nitrification enhancing agents. The study included fiveponds in and around Irving, Texas; the ponds, identified as Ponds 1-5ranged from 23,400 ft³ to 720,131 ft³. The length of this study wasseven months. One to two times per week, a surface water sample of200-300 mL was taken bank side from each pond. These samples wereanalyzed for pH, alkalinity, nitrate, phosphate, ammonia, conductivity,turbidity and E. coli spp. concentrations. Phosphate, ammonia andturbidity analysis was performed using a Hach DR890 colorimeter. E. colispp. determination was performed using specialized media for coliformgrowth (3M Petrifilm 6404) incubated at 35° C. for 48 hours.

Once per month each pond was sampled for sludge depth, clarity anddissolved oxygen (DO). These measurements were taken from a small boatat two to four locations, marked by GPS coordinates to obtainrepresentative sampling. Sludge depth was measured in inches using asludge judge; each GPS location was sampled three to four times with theaverage taken. Dissolved oxygen was measured in ppm from the bottomlayer and again at 18″ from the surface using a Hach LDO probe with aHach HQ30d meter. Clarity was determined in %/feet using a Secchi Disk,which gave an empirical measurement. Additionally, once per monthphotographs were taken at each pond at two to four locations, againmarked by GPS coordinates, to give a patron point-of-view of overallsurface conditions.

A BioAMP™ 750 climate controlled biogenerator was installed at eachlocation for daily on-site dosing of a specialized pond blend ofbacteria. Bacillus spp. spores were pelletized using a modifiedFREE-FLOW™ formula; pellets were fed into the growth vessel where theygrew in optimal conditions for 24 hours and were then dispersed directlyinto the pond. Maintenance of the BioAMP 750 had to be modified fromstandard protocol as sodium hypochlorite (bleach) is considered toxic tosurface water and not allowed by the City of Irving for use. To obtain asimilar whitening effect as the standard bleach treatment, 155 g ofsodium bicarbonate (baking soda) was used to remove excess biofilm andclean the growth vessels. In addition to monthly maintenance, thebiogenerators were monitored for any malfunctions and ability tomaintain programmed temperature despite an ambient temperaturesexceeding 100° F.

As a companion to the bacterial treatment, calcified seaweed was alsoapplied to each pond. The amount of calcified seaweed given wasdependent on volume at a ratio of 100 lbs to 1,000,000 ft³ of water. Thecalcified seaweed was dosed using water soluble packages containing aneffervescent couple and the calcified seaweed as described in U.S.patent application Ser. No. 14/689,790.

Each study pond was given the same amount of bacteria daily (30 trillionCFUs). A correlation matrix revealed that sludge depth was inverselyrelated to dose-rate. Two of the smaller ponds had the greatest observedreduction in sludge levels and clarity, as well as, the highest dailydose of bacteria at 2×10⁷ CFU/L and 7×10⁶ CFU/L respectively. Clarity asobserved, show positive effects on all ponds, regardless of size.Clarity was approximately 100% in the three smallest ponds of thisstudy. Conversely the two largest ponds only achieved clarity of 20% bythe end of this study.

A one-sided 2 sample t-test was used to evaluate if sludge levelssignificantly decreased after treatment. A p-value of 0.006 found astatistically significant average decrease of 31%. H₀: μ pre-treatment=μpost-treatment, H_(A): μ pre-treatment>μ post-treatment. This averagereduction observed equates to an average 3 inch reduction of the sludgelayer. Additionally, every pond in this study experienced a decrease insludge level when compared to pre-treatment (see Table 3).

TABLE 3 Changes in PO₄, NO₃ & Sludge by Pond Pond % Δ PO4 % Δ NO3 % ΔSludge 1 −19 −71 −45 2 −77 −100 −18 3 −40 0 −16 4 −70 −100 −37 5 −48 0−43 Average −52 −91 −31

The average observed change in E. coli spp. was a reduction of 59%. Itis important to note the full range included an increase of 145% to adecrease of 100%. Such a wide range coupled with a small sample sizemade it difficult to determine the effectiveness of the treatment on E.coli spp. concentration. Three out of the five study ponds experiencedan increase in E. coli spp. concentrations; however, the other two pondssaw a dramatic decrease; however the increase is believed to be theresult of rainwater runoff into the ponds.

The overall effect of this treatment on phosphate concentrations wasexamined, comparing pre-treatment to post-treatment levels. The data wasfound to be non-parametric and a one-sided Mann-Whitney test wasemployed to determine if phosphate concentrations significantlydecreased after treatment. H₀: μ pre-treatment=μ post-treatment, H_(A):μ pre-treatment>μ post-treatment. A p-value of 0.0000 was obtainedindicating that the overall 52% decrease in phosphate levels aftertreatment was statistically significant. Detailed examinations ofchanges in phosphate level by pond also revealed meaningful decreases.Each treated pond saw a decrease in phosphate concentrations rangingfrom 19% to 77% (see Table 3). The average of 52% reduction was similarto the 57% reduction observed in phase I of this study. This indicatesthat the increase in frequency of bacterial dosing may not be associatedwith the decrease in phosphate concentrations. Furthermore, a markeddecrease in phosphate levels was observed directly following anapplication of calcified seaweed. The first dose was administered inspring with the second given in summer after phosphate levels began torise in June. The non-chemical, eco-friendly nature of the powderedproduct offers promising results for control of phosphateconcentrations.

Similarly, nitrate levels were examined using a one-sided Mann-Whitneytest to evaluate if nitrate concentrations significantly decreased aftertreatment. H₀: μ pre-treatment=μ post-treatment, H_(A): μpre-treatment>μ post-treatment. With a p-value of 0.0000 it wasdetermined that the 91% reduction in concentration was statisticallysignificant (see Table 3). To that end, baseline nitrate concentrationswere below recommended levels so no reductions were anticipated letalone a statistically significant reduction of 91%. Furthermore, eachstudy pond that had detectable nitrate was significantly reduced tobelow detection limits of 0.01 ppm by month 4. This was a vastimprovement over the reduction observed in Phase I (˜69%) and indicatesthat the increased frequency of bacterial dosing had a direct effect onthese concentrations. Overall this study demonstrated that enhancedtreatment with bacteria and calcified seaweed increased pond health asmeasured by chemical proxies and a decrease in sludge level.

Nutrient Germinant Compositions

A nutrient germinant composition according to one preferred embodimentof the invention comprises one or more L-amino acids, D-glucose (whichincreases the binding affinity of L-amino acids for their cognatereceptors in the spore coat and is optional), D-Fructose (optional,depending on bacteria species), a biological buffer to provide theproper pH for spore germination (such as HEPES sodium salt, a phosphatebuffer, or a Tris buffer), an optional source of potassium ions (such asKCl), and an industrial preservative. In another preferred embodiment, anutrient germinant composition further comprises both D-glucose andD-fructose. It is most preferred to include a source of potassium ions,such as KCl, when both D-glucose and D-fructose are used. The use ofD-fructose, a combination of D-glucose and D-fructose, and a potassiumion source are dependent on the species of bacteria as will beunderstood by those of ordinary skill in the art. It is preferred to usea preservative that is pH compatible with the spore composition, whichhas a relatively neutral pH. According to another preferred embodiment,the nutrient spore composition also comprises spores of one or moreBacillus species and preferably one or more germination inhibitors. Anutrient germinant composition comprising spores is referred to hereinas a nutrient-spore composition, formula, or solution. Alternatively,spores may be separately added to the nutrient-germinant compositionaccording to the invention at the point-of-use. When separately added,the spores are preferably part of a spore composition or sporeformulation described herein, but other commercially available sporeproducts may also be used. According to another preferred embodiment,the nutrient germinant or nutrient spore composition is in aconcentrated form, most preferably as a concentrated liquid, and isdiluted at the point-of-use.

Preferred L-amino acids include one or more of L-alanine, L-asparagine,L-valine, or L-cysteine. The L-amino acids can be provided in the formof any suitable source, such as their pure forms and/or a hydrolysate ofsoy protein. In a further embodiment of the concentrate nutrientgerminant composition, L-amino acids can be provided as a hydrolysate ofsoy protein. When in a concentrated form, the spore compositionpreferably comprises a solution of one or more of the above mentionedL-amino acids in the weight range of about 8.9 to about 133.5 g/L, morepreferably about 13.2 to about 111.25 g/L, and most preferably about17.8 to about 89 g/L each; D-glucose (optional) and/or D-fructose(optional) in the weight range of about 18 to about 54 g/L each, morepreferably about 27 to about 45 g/L each, and most preferably about 30to about 40 g/L each; KCl (optional, as a source of potassium ions) inthe weight range of about 7.4 to about 22.2 g/L, more preferably about11.1 to about 18.5 g/L, and most preferably about 14 to about 16 g/L; abiological buffer, such as monosodium phosphate in a weight range ofabout 10 to about 36 g/L, more preferably about 15 to about 30 g/L, andmost preferably about 20 to about 24 g/L and/or disodium phosphate in aweight range of about 30 to about 90 g/L, more preferably about 21.3 toabout 75 g/L, and most preferably about 28.4 to about 60 g/L. One ormore biological buffers aid in maintaining the nutrient germinantcomposition at the proper pH for spore germination, around pH 6-8. Inaddition to or in place of the monosodium/disodium phosphate buffer, thespore composition may comprise other phosphate buffer(s), Tris base in aweight range of about 15 to about 61 g/L, more preferably about 24 toabout 43 g/L, and most preferably about 27 to about 33 g/L; or HEPESbuffer in a weight range of about 32.5 to about 97.5g/L, more preferablyabout 48.75 to about 81.25 g/L, and most preferably about 60 to about 70g/L. Optionally, monopotassium phosphate may also be used as a source ofpotassium ions, preferably in a weight range of about 13.6 to about 40.8g/L, more preferably about 20.4 to about 34 g/L, and most preferablyabout 26 to about 29 g/L. Optionally, dipotassium phosphate may also beused as a source of potassium ions, preferably in a weight range ofabout 8.7 to about 26.1 g/L, more preferably about 13 to about 21.75g/L, and most preferably about 16 to about 19 g/L. According to anotherpreferred embodiment, the amounts of KCl, monosodium phosphate, and/ordisodium phosphate can be adjusted such that the pH in the nutrientgerminant solution and/or nutrient-spore solution can be about 6, about7, or about 8.

In another preferred embodiment, the nutrient germinant compositionfurther comprises one or more industrial preservatives at a final(total) weight range of 0.8-3.3 g/L, more preferably 1.2-2.7 g/L, mostpreferably 1.6-2.2. The preservative(s) can be beneficial for long-termstorage. Suitable preservatives include, NaCl, D-alanine, potassiumsorbate, and chemical preservatives. Chemical preservatives can bepreservatives with active ingredients of methyl chloro isothiazolinone(about 1.15% to about 1.18% v/v) and methyl isothiazolinone (about0.35-0.4% v/v); preservatives with the active ingredients ofdiazolidinyl urea (about 30%), methylparaben (about 11%), andpropylparaben (about 3%); and preservatives with only the activeingredient of methylparaben; and other preservatives with the methylparaben, propylparaben, and diazolidinyl urea). Non-limiting examples ofchemical preservatives with methyl chloro isothiazolinone and methylisothiazolinone as active ingredients are Linguard ICP and KATHON™ CG(which has active ingredients comprising methyl chloro isothiazolinone,around 1.15-1.18% and methyl isothiazolinone, around 0.35-0.4%). Anon-limiting example of a chemical preservative with diazolidinyl urea,polyparaben, and methylparaben as active ingredients includes GermabenII. Where the active ingredients of the chemical preservative are methylchloro isothiazolinone and methyl isothiazolinone, the chemicalpreservative can be included in a concentrated nutrient solution atabout 0.8 to about 3.3 g/L, more preferably from about 1.2 to about 2.7g/L, and most preferably from about 1.6 to about 2.2 g/L. Where theactive ingredient(s) of the chemical preservative is diazolidinyl urea,methylparaben, and/or propylparaben, the chemical preservative can beincluded in a concentrated nutrient solution at about 0.3 to about 1%(wt/wt). In some aspects, the amount of a chemical preservative havingdiazolidinyl urea, methylparaben, and propylparaben can be included inthe nutrient formulation at about 10 g/L. In the case of methylparaben,the preservative can be included in a concentrated nutrient solution atabout 0.27 to about 1.89 g/L, more preferably from about 0.81 to about1.35 g/L, and most preferably from about 1.0 to about 1.18 g/L.According to another preferred embodiment, where the nutrientformulation can be used to generate a nutrient-spore formulationeffective for aquaculture applications involving shrimp, or othershellfish, the preservative can include an amount of methylparaben andpotassium sorbate. According to another preferred embodiment, a nutrientgerminant solution can be used to generate a nutrient-spore formulationeffect for plants and/or waste water, the nutrient-spore formulation caninclude an amount of Linguard ICP or KATHON™ CG.

According to yet another preferred embodiment, a nutrient germinantcomposition may further optionally comprise an osmoprotectant compound.Ectoine, a natural osmoprotectant produced by some species of bacteria,may be included in one preferred embodiment. The amount of ectoine(optional) in a concentrated nutrient germinant composition can rangefrom about 0.625 to about 4.375 g/L, more preferably from about1.875-3.125 g/L, and most preferably in an amount around 2-3 g/L.According to another preferred embodiment, a nutrient germinantcomposition may further comprise other standard ingredients including,but not limited to, surfactants that aid in the dispersal of activeingredients, additional preservatives ensure the shelf-life of the sporecomposition, buffers, diluents, and/or other ingredients that aretypically included in a nutrient formulation and/or spore formulation.

The amounts of the above ingredients are important aspects of theinvention because higher concentrations would render some ingredientsinsoluble and lower concentrations would be ineffective at germinatingspores.

According to another preferred embodiment, a nutrient-germinantconcentrate composition according to embodiments of the invention is inconcentrated form and is diluted to a working solution in water, a sporecomposition, or any other appropriate diluent, or a combination thereofprior to germination at a point-of-use as described further below.According to various preferred embodiments, a working nutrient germinantsolution may be made by diluting a concentrated nutrient germinantcomposition according to a preferred embodiment herein with water orother suitable diluent in a ratio between 0.01% to 50% (v/v)concentrated nutrient germinant composition to diluent, but otheramounts may also be used. The concentrated nutrient germinantcompositions according to the invention may diluted anywhere from 2 to1×10⁶ fold or any range or value therein to produce a working nutrientgerminant solution. Most preferably, dilution is in a range from about0.1 to about 10% of the concentrate and the balance water or othersuitable diluent. The amounts of the above described ingredients presentin a working nutrient solution (a diluted solution from a concentratedformula) may be calculated based on the dilution factor and theconcentrated amounts described above.

The use of a concentrated nutrient germinant composition reducesshipping, storage, and packaging costs and makes dosing of the sporecomposition at the point-of-use easier. Most preferably, theconcentrated nutrient germinant composition is in a liquid form, whichis easier and faster to mix with diluent at the point-of-use, but solidforms such as pellets or bricks or powder may also be used. Theinclusion of a general, industrial preservative in the nutrientgerminant composition aids in long-term storage.

Most preferably, all ingredients in nutrient germinant compositionsaccording to the invention or used with methods of the invention meetU.S. federal GRAS standards.

Nutrient Spore Compositions

According to another preferred embodiment, the composition is anutrient-spore composition comprising ingredients described above for anutrient germinant composition and spores pre-mixed together. Accordingto one preferred embodiment, a nutrient spore composition preferablycomprises 10% to 90% by weight of one or more Bacillus spores or a sporeblend (comprising 40-60% spore powder with one or more Bacillus speciesand 60-40% salt). According to another preferred embodiment, a nutrientspore composition comprises around 5% by weight of one or more Bacillusspores or a spore blend. The total concentration of spores in thenutrient spore composition can range from about 1×10⁵ CFU/mL or spores/gto 1×10¹⁴ CFU/mL or spores/g or any specific concentration or rangetherein.

A nutrient-spore composition preferably also comprises one or moregermination inhibitors and/or preservatives. Preferred germinationinhibitors or preservatives for a concentrated nutrient sporecomposition include NaCl at a relatively high concentration ranging fromabout 29 to about 117 g/L, more preferably from about 43 to about 88g/L, and most preferably from about 52 to about 71 g/L; and/or D-alaninein an amount ranging from about 8 to about 116 g/L, more preferably fromabout 26 to about 89 g/L, and most preferably from about 40 to about 50g/L; and/or potassium sorbate in an amount ranging from about 1.25 toabout 8.75 g/L, more preferably from about 3.75 to about 6.25 g/L, andmost preferably from about 4.5 to about 5.5 g. Other chemicalpreservatives described above with preferred nutrient germinantcomposition may also be used with nutrient spore compositions accordingto the invention. These germination inhibitors or preservatives maintainthe spores in an inactive state and prevent premature germination of thespores prior to their dilution and activation at the point-of-use. Theuse of germination inhibitors is particularly preferred when the sporecomposition according to this embodiment is used with the preferredmethod of the invention, where germination occurs at the point-of-use.When spores are included, the amounts of other ingredients for thenutrient germinant composition described above (such as L-amino acids,biological buffers, etc.) make up the balance of the spore compositionreduced proportionally to correspond to the preferred ranges describedabove. Preferred nutrient spore compositions are also in concentratedform and diluted to a working solution at a point-of-use as describedabove with respect to nutrient germinant composition and furtherdescribed below.

The preferred Bacillus spores for use in a nutrient spore compositionaccording to preferred embodiments of the invention include thefollowing species: Bacillus licheniformis, Bacillus subtilis, Bacillusamyloliquiefaciens, Bacillus polymyxa, Bacillus thuringiensis, Bacillusmegaterium, Bacillus coagulans, Bacillus lentus, Bacillus clausii,Bacillus circulans, Bacillus firmus, Bacillus lactis, Bacilluslaterosporus, Bacillus laevolacticus, Bacillus polymyxa, Bacilluspumilus, Bacillus simplex, and Bacillus sphaericus. Other Bacillus sporespecies may also be used as will be understood by those of ordinaryskill in the art. Preferably, a nutrient spore composition comprises 1to 20 or more species of Bacillus, more preferably between 3 to 12Bacillus species. According to another preferred embodiment, a nutrientspore composition comprises 3 strains of Bacillus bacteria, mostpreferably 2 strains of the Bacillus bacteria can each be a strain ofthe species Bacillus licheniformis and the third strain is a species ofBacillus subtilis. According to another preferred embodiment, the sporesin a spore blend comprise about 80% Bacillus licheniformis (40% of eachstrain) and 20% Bacillus subtilis.

In another preferred embodiment, a nutrient-spore composition for use asa probiotic comprises one or more Bacillus strains that are probiotic innature in that they aid in the breakdown of nutrients in the digestivetract of the consumer. The strains preferably produce one or more of thefollowing enzymes: proteases to hydrolyze proteins, amylases tohydrolyze starches and other carbohydrates, lipases to hydrolyze fats,glycosidases to assist in the hydrolysis of glycosidic bonds in complexsugars and to assist in degradation of cellulose, cellulases to degradecellulose to glucose, esterase which is a lipase-like enzyme, andxylanases that degrade xylan, a polysaccharide found in plant cellwalls. Bacillius strains that produce these enzymes are well known inthe art.

According to another preferred embodiment, a nutrient spore compositionis in a concentrated form and is diluted with to a working solution inwater or any other appropriate diluent, or a combination thereof, priorto germination at a point-of-use as described further below. Accordingto various preferred embodiments, a working nutrient spore solution maybe made by diluting a concentrated nutrient spore composition accordingto a preferred embodiment herein with water or other suitable diluent ina ratio between 0.01% to 50% (v/v) concentrated nutrient germinantcomposition to diluent, but other amounts may also be used. Theconcentrated nutrient spore compositions according to the invention maydiluted anywhere from 2 to 1×10¹³ fold or any range or value therein toproduce a working nutrient germinant solution. Most preferably, dilutionis in a range from about 0.1 to about 10% of the concentrate and thebalance water or other suitable diluent. The amounts of the abovedescribed ingredients (such as L-amino acids and germination inhibitors)present in a working nutrient solution (a diluted solution from aconcentrated formula) may be calculated based on the dilution factor andthe concentrated amounts described above

Most preferably, all ingredients in nutrient spore compositionsaccording to the invention or used with methods of the invention meetU.S. federal GRAS standards.

Spore Compositions

A probiotic spore composition according to one preferred embodiment ofthe invention comprises one or more bacterial species, an optionalsurfactant, a thickener, and optionally one or more acidifiers, acids orsalts or acids to act as a preservative. According to another preferredembodiment, a spore composition further comprises one or moreprebiotics, to the extent the thickener is not also a prebiotic, or inaddition to any thickener that is a prebiotic. According to anotherpreferred embodiment, a spore composition further comprises one or morewater activity reducers. Most preferably, the spore compositionsaccording to the invention comprise various species of suspendedprobiotic spores, as described in more detail below. The use of thesespecies in spore form increases the stability of the probiotics in theharsh environmental conditions that may be found near aquacultureapplication sites. The total concentration of spores in the sporecomposition can range from about 1×10⁵ CFU/mL or spores/g to 1×10¹⁴CFU/mL or spores/g or any specific concentration or range therein.

A suitable thickener is included in the spore composition according topreferred embodiments. The thickener is preferably one that does notseparate or degrade at varying temperatures typically found innon-climate controlled aquaculture environments. The thickener aids instabilizing the suspension so the bacterial mixture remains homogenousand dispersed through a volume of the spore composition and does notsettle out of the suspension. When used with an incubation system andaquaculture treatment methods according to preferred embodiments of theinvention described herein, this ensures that the concentration ofprobiotic materials is evenly distributed throughout the container sothat the dosage of spores delivered to an incubator remains consistentor relatively consistent (depending on the specific delivery method andcontrol mechanism used) throughout a treatment cycle.

The most preferred thickener is xanthan gum, which is a polysaccharidecomposed of pentasaccharide repeat units of glucose, mannose, andglurcuronic acid and a known prebiotic. Unlike some other gums, xanthangum is very stable under a wide range of temperatures and pH. Anotherpreferred thickener is acacia gum, which is also a known prebiotic.Other preferred thickeners include locust bean gum, guar gum and gumarabic, which are also believed to be prebiotics. In addition toprebiotic benefits, these fibers do not bind to minerals and vitamins,and therefore, do not restrict or interfere with their absorption andmay even improve absorption of certain minerals, such as calcium, byaquatic species. Other thickeners that are not considered prebiotics mayalso be used.

Preferred embodiments may optionally include one or more prebiotics,which are preferably used if the thickener used is not a prebiotic butmay also be used in addition to a prebiotic thickener. Prebiotics areclassified as disaccharides, oligosaccharides and polysaccharides, andcan include Inulin, Oligofructose, Fructo-oligosaccharides (FOS),Galacto-oligosaccharide (GOS), trans-Glacto-Oligosaccharides (TOS) andShort-Chain Fructo-oligosaccharides (scFOS), soy Fructo-oligosaccharide(soyFOS), Gluco-oligosaccharides, Glyco-oligosaccharides, Lactitol,Malto-oligosaccharides, Xylo-oligosaccharides, Stachyose, Lactulose,Raffinose. Mannan-oligosaccharide (MOS) are prebiotics may not enrichprobiotic bacterial populations, but will bind with and remove pathogensfrom the intestinal tract and are believed to stimulate the immunesystem.

Preferred embodiments also preferably include one or more acidifiers,acids, or salts of acids to act as a preservative or to acidify thespore composition. Preferred preservatives are acetic acid, citric acid,fumaric acid, propionic acid, sodium propionate, calcium propionate,formic acid, sodium formate, benzoic acid, sodium benzoate, sorbic acid,potassium sorbate, and calcium sorbate. Other known preservatives,preferably generally regarded as safe (GRAS) food preservatives, mayalso be used. Preferably, the pH of the spore composition is betweenabout 4.0 and 7.0. More preferably it is between about 4.0 and 5.5 andmost preferably around 4.5 to prevent premature germination of thespores prior to use or addition to an incubator as described below.Reducing the pH of the spore composition may also have antimicrobialactivity with respect to yeast, molds, and pathogenic bacteria.

One or more water activity reducers, such as sodium chloride, potassiumchloride, or corn syrup (a 70% solution of corn syrup), are optionallyincluded in the spore composition according either preferred embodiment.The water activity reducer aids in inhibiting microorganism growth, sothat the bacterial spores do not prematurely germinate while the sporecomposition is being stored prior to the time it is discharged to thepoint of consumption by the animals or plants to be treated or point ofuse in discharging to a growing pond. They also aid in inhibiting growthof contamination microorganisms

The optional surfactant is preferably one that is safe for ingestion byanimals, although other surfactants may be used with other applications,such as delivery to plants. Most preferably, the surfactant isPolysorbate 80. Although any GRAS or AAFCO approved surfactants oremulsifiers may be used with either embodiment, there are concerns thatsome animals may not tolerate all approved surfactants well. Because thebenefits of the surfactant in stabilizing the suspension so thebacterial mixture remains homogenous and does not settle out may also beachieved by the use of the thickener, it is not necessary to add thesurfactant. If a surfactant is used in the spore composition accordingto this second embodiment, it is preferably used in about the sameweight percentage range as in the first embodiment.

Preferred bacteria for use with a spore composition according to theinvention are the same as those described above for a preferred nutrientspore composition. Most preferably, the bacterial species used in aspore composition are one or more species from the Bacillus genus. Themost preferred species for the probiotic bacteria include the following:Bacillus pumilus, Bacillus licheniformis, Bacillus amylophilus, Bacillussubtilis, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillusclausii, Bacillus firmus, Bacillus megaterium, Bacillus mesentericus,Bacillus subtilis var. natto, or Bacillus toyonensis, but any Bacillusspecies approved as a probiotic in the country of use may also be used.It is preferred that the bacteria are in spore form, as the spore formis more stable to environmental fluctuations, such as ambienttemperature changes. Most preferably, the spores used in the sporecompositions according to the invention are a dry powder blend thatcomprises around 40-60% salt (table salt) and 60-40% bacterial spores.The spores are preferably spray-dried from a liquid fermentationconcentrate. Salt is used to dilute the pure spray-dried spore powder toa standard spore count in the final spore powder blend. Duringproduction fermentation, different Bacillus strains will grow atdifferent rates, resulting in varying final count numbers for thefermentation batch liquor. The fermentation liquor is centrifuged toconcentrate the spores in the liquor. Then, the concentrated liquor isspray-dried which results in a powder containing only Bacillus spores.The addition of salt to the spray-dried Bacillus spore powder aids instandardizing the spore blend count per gram from batch to batch. Otherforms of bacterial spores or spore blends may also be used. Mostpreferably, the dry spore blend is pre-mixed with a portion of the waterused in the spore composition, around 3-30% of the total water, and theresulting bacteria spore solution is added to the other ingredients,including the remaining water. This aids in dispersing the bacteriaspores throughout the spore composition.

A probiotic spore composition according to a first preferred embodimentof the invention preferably comprises bacterial spores that provide 10⁸cfu/ml of the spore suspension (most preferably around 1.0×10⁸ to around3.0×10⁸ cfu/ml of spore composition, which, when diluted in growing pondwater provides approximately 10¹ to 104 cfu/ml pond water), 0.00005 to3.0% surfactant, and 0.002 to 5.0% thickener, and optionally the about0.01 to 2.0% of one or more acids or salts of acids as a preservative,all percentages by weight of the spore composition. A probiotic sporecomposition according to another preferred embodiment of the inventioncomprises bacterial spores that provide 10⁹ cfu/ml of the sporesuspension (which, when diluted in pond water provides approximately 10¹to 10⁴ cfu/ml pond water), about 0.1 to 5.0% thickener (preferably onethat also acts as a prebiotic), about 0.05-0.5% of one or morepreservatives, optionally about 0.1-20% of one or more water activityreducers, and optionally 0.1-20% of one or more acidifiers, allpercentages by weight of the composition. The balance of the sporecomposition in both preferred embodiments is water and the percentagesherein are by weight. It is preferred to use deionized or distilledwater, to remove salts or outside bacteria, but tap water or othersources of water may also be used.

According to another preferred embodiment, a spore composition comprisesaround 1% to 10% of a bacteria spore blend containing salt and one ormore of Bacillus pumilus, Bacillus licheniformis, Bacillus amylophilus,Bacillus subtilis, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus megaterium, Bacillus mesentericus, Bacillus subtilisvar. natto, or Bacillus toyonensis in spore form; around 0.3% to 1%total of one or more acids or salts of acids; around 0.2% to 0.5% of athickener; around 0.1-0.3% sodium chloride, potassium chloride, or acombination thereof; around 0.00005% to 3.0% of a surfactant; and 86.2%to 98.4% water. According to another preferred embodiment, a sporecomposition comprises around 0.01% to 10% of the bacteria spore blend;around 0.1-0.33% sorbic acid, its salt, or a combination thereof; around0.1-0.34% citric acid, its salt, or a combination thereof; around0.1-0.33% benzoic acid, its salt, or a combination thereof; around0.2-0.5% xanthan gum; around 0.00005% to 3.0% of a surfactant; andaround 0.1-0.3% sodium chloride, potassium chloride, or a combinationthereof, all percentages by weight of the composition. According to yetanother preferred embodiment, a spore composition comprises around 5%bacteria spores or a spore blend; around 0.25% thickener; around 0.3%total of one or more acids or salts of acids; around 0.1% surfactant;around 0.2% sodium chloride, potassium chloride, or a combinationthereof (in addition to any salt in the spore blend); and water.According to another preferred embodiment, the acids or salts of acidsare one or more of potassium sorbate, sodium benzoate, and citric acidanhydrous.

Several examples of probiotic spore compositions according preferredembodiments of the invention were made and tested for differentparameters. These spore compositions are set forth in Table 4 below.

TABLE 4 Ingredient/ Formula No. 1 2 3 4 5 6 7 8 Potassium 0.33% 0.33%0.1% 0.1%  0.1% 0.1% 0.1% 0.1% Sorbate Citric Acid 0.34% 0.34% 0.1% 0.1% 5.0% 0.1% 0.1% 0.1% Sodium 0.33% 0.33% 0.1% 0.1%  0.1% 0.1% 0.1% 0.1%Benzoate Benzoic — — — 0.1% — 0.1% 0.1% — Acid Sorbic — — — 0.1% — —0.1% — Acid Sodium — — — — 10.0% 0.1% — — Propionate Xanthan 0.2%  0.2% 0.2% 0.3%  0.4% 0.4% 0.5% 0.5% Gum Sodium 0.2%  0.2%  — 0.2% — 0.2% 0.1%0.2% Chloride Potassium — — — — — — 0.1% 0.1% Chloride Spore 0.1%  0.1% 0.1% 0.1%  0.1% 0.1% 0.1% 0.1% Blend

The balance of each spore composition is water (around 1 L in thesesamples). Deionized water was used in each spore composition, exceptspore composition No. 1, which used tap water. The percentages indicatedare by weight. Each formula was targeted to have a pH between about 4.0and 5.5, but some formulas were found to have actual pH values far lessthan expected. Formula No. 1 was targeted to have a pH between 5.0 and5.5, but its actual pH was around 2.1-2.3, which is too low and may beharmful to the spores, create stability issues with packaging, and besubject to more restrictive transportation regulations. Formula No. 1also exhibited weak thickening. Formula No. 2 is the same as No. 1,except the source of water is different. Formula No. 2 had an actual pHof around 2.2-2.3 and also exhibited weak thickening. The amount ofacids and salts of acids in Formula No. 3 was decreased to raise the pHand to determine if the thickness improved while using the same amountof thickener as in Nos. 1 and 2. While Formula No. 3 was an improvementover Nos. 1 and 2, it still exhibited weak thickening and its actual pHwas 6.6, over the target value range. Additional acids were added toFormula No. 4 to lower the pH and additional thickener was added.Formula No. 4 had improved thickening, but further improvements inthickening would be beneficial. The amount of acid in Formula No. 5 wassubstantially increased, which resulted in an actual pH of around 1.0.The amount of acid in Formula No. 6 was decreased and the thickenerincreased, which resulted in a spore composition that was too thick todrop. Formula No. 7 increased the thickener and amount of water activityreducers, but exhibited issues with mixing of benzoic acid and sorbicacid. The benzoic acid and sorbic acid were removed in Formula No. 8.Formula Nos. 1-7 provided 2×10¹¹ cfu/gm and No. 8 provided 1×10¹¹ cfu/gmbacteria spores. Of these sample formulas, No. 8 is the most preferredas it exhibited adequate thickening and had an actual pH of around4.5+/−0.2, and used less spore blend.

It is preferred that the spore compositions according the embodiments ofthe invention use around 0.01% to around 0.3% bacteria spore blend andmore preferably between about 0.03% to 0.1% bacteria spore blend. Areduction in the amount of spore blend used substantially reduces thecosts of the spore composition. Depending on the end use application,differing amounts of spore blend may be used in the spore compositionsaccording to the invention. For example, smaller percentages of sporeblend may be used in the spore compositions for use with chickens,whereas larger percentages would be used in spore composition for usewith pigs.

A spore composition according to formula No. 8 was tested for shelf-lifeat various temperatures. Samples of Formula No. 8 were sealed in aplastic bag, such as one used in a preferred delivery system asdescribed below, and stored for two months at temperatures around 4-8°C. (39-46° F.), 30° C. (86° F.), and 35° C. (95° F.) to simulate typicaltemperature ranges in which the probiotic spore composition may bestored and used in agricultural settings. At the end of the first monthof the storage period, each sample was observed and tested. All threesamples had a pH of around 4.5 and there was no settling, layering orchange of appearance in any of the three samples, indicating that thebacteria spores remained suspended and dispersed throughout the sporecomposition during the storage period. None of the samples contained anyfungal contamination or gram-negative bacteria contamination. At timecount zero (when the samples were initially stored), each samplecontained bacteria spores of around 2.12×10⁸ cfu/mL. At the end of theone month storage period, the samples contained bacteria spores ofaround 2.09×10⁸ cfu/mL spore suspension (lowest temperature sample),1.99×10⁸ cfu/mL (middle temperature sample), and 2.15 10⁸ cfu/mL (hightemperature sample). The bacteria counts are somewhat variable indifferent samples, especially thickened samples; however, these areconsidered to be comparable counts. Each sample was tested again aftertwo months in storage. The samples contained bacteria spores of around2.08×10⁸ cfu/ml (lowest temperature sample); 2.01×10⁸ cfu/ml (middletemperature sample); and 2.0×10⁸ cfu/ml (high temperature sample). Thetarget shelf life is around 2×10⁸ cfu/ml spore suspension, so thesamples are within the targeted shelf life after two months of storage.These test results demonstrate that probiotic spore compositionsaccording to a preferred embodiment of the invention are stable over arange of temperatures, with the bacteria spores remaining viable,suspended, and dispersed throughout the spore composition. The sporeblend (40-60% spore powder and 60-40% salt) used in each sample formulawas the same, providing at least around 2×10¹¹ spores/gram. The sporespecies in the blend were multiple Bacillus subtilis and Bacilluslicheniformis strains. The spore blend powder was premixed with 100 mLof water with stirring for 30 minutes prior to adding to the otheringredients. Premixing with water aids in mixing the spore blend withthe other ingredients and dispersing the spores throughout the sporecomposition.

Although it is preferred to use probiotic spore compositions comprisingone or more Bacillus species as according to the spore compositions ofthe invention, the methods of the invention may be used with sporecompositions comprising other bacteria genera and other species. Forexample, one or more species from the following genera: Bacillus,Bacteriodes, Bifidobacterium, Pediococcus, Enterococcus, Lactobacillus,and Propionibacterium (including Bacillus pumilus, Bacilluslicheniformis, Bacillus amylophilus, Bacillus subtilis, Bacillusamyloliquefaciens, Bacillus clausii, Bacillus coagulans, Bacillusfirmus, Bacillus megaterium, Bacillus mesentericus, Bacillus subtilisvar. natto, or Bacillus toyonensis Bacteriodes ruminocola, Bacteriodesruminocola, Bacterioides suis, Bifidobacterium adolescentis,Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacteriuminfantis, Bifidobacterium longum, Bifidobacterium thermophilum,Pediococcus acidilacticii, Pediococcus cerevisiae, Pediococcuspentosaceus, Enterococcus cremoris, Enterococcus diacetylactis,Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis,Enterococcus thermophilus, Lactobacillus delbruekii, Lactobacillusfermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillusplantarum, Lactobacillus reuteri, Lactobacillus brevis, Lactobacillusbuchneri, Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillusfarciminis, Lactobacillus cellobiosus, Lactobacillus curvatus,Propionibacterium acidipropionici, Propionibacterium freudenreichii,Propionibacterium shermanii) and/or one or more of the followingspecies: Leuconostoc mesenteroides, Megasphaera elsdennii may be usedwith compositions and method of the invention.

Spore compositions may also be in a concentrated form, using less waterwith a proportional increase in the amounts of other ingredients asdescribed above. Such concentrated spore compositions may be diluted atthe point-of-use with a nutrient germinant composition, water, othersuitable diluent or a combination thereof prior to germination. Mostpreferably, all ingredients in spore compositions according to theinvention or used with methods of the invention meet U.S. federal GRASstandards.

Methods of Germination

According to one preferred embodiment, a method of germinating spores ata point-of-use according to the invention comprises providing nutrientsand spores (preferably providing a nutrient germinant composition and aspore composition or providing a nutrient spore composition according tothe invention, but other commercially available products containingspores and nutrients, together or separately, may be used) and heatingthem to an elevated temperature or range of temperatures and maintainingthem at that temperature or within that range for a period of time(incubation period) to allow germination at a point-of-use location neara point-of-consumption. Heating during the incubation period takes placein a single step with both the nutrients and spores together. The methodalso preferably comprises the step of dispensing the germinated sporesto an aquaculture application as previously discussed. Preferably, thenutrient germinant composition and spore composition (or nutrient sporecomposition) is heated to a temperature in a range of 35-55° C., morepreferably in the range of 38-50° C., and most preferably in the rangeof 41° C. to 44° C. The incubation period can vary depending on theend-use application. For a probiotic application, where the aquaticspecies with a digestive system (e.g. fish or eels) will ingest thebacteria, it is preferred that the incubation period lasts no longerthan 10 minutes. Most preferably, in a probiotic application, theincubation period is between 2-5 minutes. In this way, spores arereleased to the growing pond before the spores have fully germinated andstand a better chance of surviving through to aquatic species'intestinal tract where they are most beneficial. For treating the waterin an aquaculture application, such as may be done with a shrimpaquaculture application, the preferred incubation time is at least onehour to allow the spores to fully germinate before discharging to thewater, more preferably 4 to 6 hours, to allow the bacteria to becomevegetative before discharging to the water. Most preferably, a nutrientgerminant composition and a spore composition (or a nutrient sporecomposition), preferably in accordance with an embodiment of theinvention discussed herein, are added to an incubator to incubate thespores at the above preferred temperature ranges and durations toproduce a bacteria solution having bacteria in a vegetative state. Theincubation may be in an air incubator, a water incubator, or any otherchamber that provides even, constant heat at the given temperaturerange. The bacteria solution is then discharged to an aquacultureapplication as previously discussed. If a concentrated nutrientgerminant composition is used, diluent water is preferably added to theincubator with the nutrient germinant composition.

Various nutrient germinant compositions according to preferredembodiments of the invention were tested according to preferred methodsof the invention. The compositions, methods, and results are describedbelow.

EXAMPLE 1—To germinate spores, FreeFlow LF-88 Probiotic (spore liquidformula commercially available from NCH Corporation) was added to 1 mLof tap water at a final concentration of approx. 1×10⁹ CFU/mL, where CFUstands for colony forming unit. A nutrient germinant concentratecomposition according to a preferred embodiment of the inventioncomprising L-alanine (89 g/L), monosodium phosphate (20 g/L), disodiumphosphate (60 g/L), and Linguard CP (1.6 g/L total) was added to thewater and bacteria mixture to provide a 4% final concentration ofnutrient-germinant composition by total weight of the mixture. Forcomparison, negative control reactions were prepared with the sameamount of FreeFlow LF-88 Probiotic and water, but without adding thenutrient germinant concentrate composition. Both mixtures (germinant andnegative control without the nutrient-germinant composition) wereblended and incubated for 60 minutes in a pre-incubated heat block setto 42° C. or at ambient room temperature (around 23° C.).

Spores from each reaction were observed using phase contrast microscopy.Slides were prepared using standard procedures. Spores were viewed on anOlympus BX41 microscope (100× oil emersion objective) and imaged usingan Olympus UC30 camera controlled by the cellSens Dimension softwarepackage.

Images were taken and germinated spores were counted as a percentage ofthe total spores in the field. A total of 10 representative images wereanalyzed for each condition (test mixture). Germinated spores lose theirrefractivity due to the influx of water and are phase-dark whilenon-germinated spores are phase-bright.

FIG. 7 shows representative images from these tests. Image A representsspores that had been germinated using a nutrient-germinant compositionand heated during the incubation period at 42° C. according to apreferred spore composition and preferred method of the invention. Thedarker spots show germinated spores, the lighter spots shownon-germinated spores. Image B represents spores that had beengerminated using a nutrient-germinant composition according to apreferred embodiment of the invention, but were incubated at ambienttemperature (23° C.). Images C-D represent control spores that had notbeen treated with a nutrient germinant composition according to theinvention, one having been incubated at 42° C. and one incubated atambient temperature (23° C.).

As can be seen in FIG. 7, the “A” image shows significantly moregerminated spores (dark spots) than the other images. Spores incubatedwith a nutrient-germinant composition according to a preferredembodiment invention in combination with a germination method accordingto a preferred embodiment of the invention show an apparent germinationefficiency of 96.8% (Example 1, FIG. 7A). Control spores that had beenincubated with a nutrient-germinant composition according to a preferredembodiment of the invention, but without using a germination methodaccording to a preferred embodiment of the invention showed an apparentgermination efficiency of 2.3% (Example 1, FIG. 7B). Similarly, sporesthat had not been incubated with a nutrient-germinant compositionaccording to the invention showed an apparent activation efficiency of1.2% and 2.6% at 42° C. and 23° C., respectively (Example 1, FIGS. 7Cand 7D). Germinated spores in the samples not treated by preferredembodiments of the present method represent the small percentage ofspores already germinated in the FreeFlow LF-88 Probiotic solution. Thisexample demonstrates that spore germination is significantly increasedwhen a nutrient-germinant composition and incubation method according topreferred embodiments of the invention are used together.

EXAMPLE 2—Another set of incubation tests were run using the same testmixture/incubation method (using the same nutrient-germinant compositionand heated incubation, “Treated Spores, 42° C.”) and controlmixture/incubation method (no nutrient-germinant composition and noheat, “Non-treated Spores, 23° C.”) as described above in Example 1 wererepeated, but different tests were run to compare the efficacy of thetest mixture according to preferred embodiments of the invention ascompared to the control mixture. Additionally, two other mixtures weretested—one in which the nutrient-germinant composition of Example 1 wasused but without heat (“Treated Spores, 23° C.”) and one in which nonutrient-germinant was used but the spores were heated (“Non-TreatedSpores, 42° C.”). Briefly, spores were incubated at 42° C. or 23° C. for1 hour with or without treatment with a preferred nutrient-germinantcomposition. After incubation, the spores from 1 mL of each reactionwere pelleted at 14K RPM for 3 min at 23° C. and resuspended in 1 mL ofButterfield's buffer. Approx. 6×10⁵ CFUs (0.02 mL) were added to 0.980mL of Davis minimal media (containing 3% glucose as a carbon source andtrace elements) with an excess of D-alanine. D-alanine is a potentinhibitor of L-amino acid-mediated germination.

Approx. 1.2×10⁵ CFUs were added to each of four wells of a PreSensOxoPlate. PreSens OxoPlates use optical oxygen sensors to fluorescentlymeasure the oxygen content of the sample using two filter pairs(excitation: 540 nm, emission: 650 nm and excitation: 540, emission: 590nm). Controls were performed as described by the manufacturer andmeasurements were taken on a BioTek 800FLx fluorescence plate reader.Time points were taken every 10 minutes for 24 hours at 37° C. withcontinual shaking and data was processed to determine the partialpressure of oxygen (pO₂) using the following formula:

pO ₂=100*[(K ₀ /IR)−1(K ₀ /K ₁₀₀)−1]

Spores that have germinated and continue to divide and grow asvegetative cells consume oxygen as part of their metabolic growth.Oxygen consumption is represented by a drop in pO₂. Presumably, thegrowth that is observed is due to the outgrowth and vegetative growth ofspores germinated by the present invention. The pO₂ levels for thesetests are shown in FIG. 8.

As shown in FIG. 8, incubation with the test mixture and methodaccording to preferred embodiments of the invention (Treated spores 42°C., using both the nutrient-germinant composition and heating) resultedin spores that began vegetative growth 4 hours faster than the controlspore mixtures that had not been treated or heated according topreferred embodiments of the invention or had been either treated with anutrient-germinant composition or heated, but not both together. Thegrowth seen in the control experiments presumably represents the approx.2% of germinated spores present in FreeFlow LF-88 Probiotic (see EXAMPLE1). This example further indicates that spore germination issignificantly increased when a nutrient-germinant composition andincubation method according to preferred embodiments of the inventionare used.

EXAMPLE 3—Another set of incubation tests were run using a similar testand control mixture and incubation method as described above inExample 1. Briefly, LF-88 was added to 10 mLs of distilled water at afinal concentration of approx. 10⁸ CFU/mL. Samples were incubated atvarious temperatures to show the efficacy of the test method accordingto preferred embodiments of the invention as compared to the controlmixture. Reactions were prepared with the nutrient-germinant compositiondescribed in Example 1 (“Treated spores” in FIG. 9) and incubated at 23°C. (ambient temperature, no heating), 32° C., 42° C., or 60° C. Acontrol reaction was incubated at ambient room temperature with nonutrient-germinant composition. After one hour of incubation, 1 mL ofeach reaction was pelleted at 14K RPM for 3 min at 23° C. andresuspended in Butterfield's buffer. Approx. 6×10⁵ CFUs (0.02 mL) wereadded to 0.980 mL of Davis minimal media (containing 3% glucose as acarbon source and trace elements) with an excess of D-alanine.

Approx. 1.2×10⁵ CFUs were added to each of four wells of a PreSensOxoPlate. Controls were performed as described by the manufacturer andmeasurements were taken on a BioTek 800FLx fluorescence plate readerusing two filter pairs (excitation: 540 nm, emission: 650 nm andexcitation: 540, emission: 590 nm). Time points were taken every 10minutes for 24 hours at 37° C. with continual shaking and data wasprocessed to determine the partial pressure of oxygen (pO₂). The pO₂levels for these tests are shown in FIG. 9.

As shown in FIG. 9, incubation using a nutrient-germinant compositionand heating according to preferred embodiments of the invention resultedin spores that began vegetative growth hours before the control. Sporestreated with the nutrient-germinant composition but not heated arecomparable to the control mixture. Spores treated with thenutrient-germinant composition that were incubated at a temperaturebelow the preferred range of range of 35-55° C. according to oneembodiment of the invention (represented by the “Treated spores 32° C.”curve) begin vegetative growth faster than control experiments, but notas fast as spores treated at elevated temperatures within the preferredranges according to the invention. Spores treated with anutrient-germinant composition and incubated at a temperature within themost preferred range of 41° C. to 44° C. according to an embodiment ofthe invention showed the best results, being the first to beginvegetative growth and beginning growth 4 hours faster than the control.As seen in previous examples, growth seen in the no-treatment controlexperiment presumably represents the approx. 2% of germinated sporespresent in FreeFlow LF-88 Probiotic (see EXAMPLE 1). This examplefurther indicates that spore germination is significantly increased whena nutrient-germinant composition and incubation method according topreferred embodiments of the invention are used.

Aquaculture Study Using Nutrient Germinant Composition and SporeComposition.

Another study was conducted to evaluate the use of preferred nutrientgerminant and spore compositions with a preferred germination andaquaculture treatment method according to the invention. Three aquariumswere used for this study as representative aquaculture applications.Each held 55 gallons of water and 25 Malaysian prawns to mimic stockingdensities of commercial shrimp farms. Each aquarium contained the sametype of netting and substrate composed of PVC pipe provided for shrimphabitation and resting. All aquariums were lined with Caribbean livesand to discourage algal growth, reduce nitrates, help buffer theaquarium system, and ensure safer aquarium cycling. Aeration stones wereused in all three aquariums to improve biological filtration andincrease dissolved oxygen content for shrimp and beneficial bacteria.All three aquariums used the same type of filter and filters were rinsedoff, as needed, and reused. All three aquariums were refilled withdeionized (DI) water as needed. DI water was used to control mineralcontent of the water.

When large amounts of water needed to be removed from an aquarium, thesame amount of water was removed from all aquariums and replaced withthe same amount of DI water. Calcium carbonate was used in aquariums 2and 3 for water replacements to mimic the use of a Pond Powder, such asECOCharger™ Pond Powder available from NCH Life Science. When water wasreplaced in aquariums 2 or 3, about 0.5 g of calcium carbonate was addedto tank water. About 1 mL of an incubated or activated bacteria solutionwas applied once daily Monday-Friday to aquarium 3. Aquarium 1 was thecontrol aquarium. Briefly, 20 μL of a starting spore solution(containing about 10¹⁰ CFU/mL) was mixed with 20 μL of a startingconcentrated nutrient solution and 960 μL of water to form a workingsolution that contained about 2×10⁸ CFU/mL spores (Table 6). Thestarting spore solution contained about 10¹⁰ CFU/mL spores from a sporeblend. The spore blend contained 3 strains of Bacillus bacteria: 2strains were each a strain of the species Bacillus licheniformis and thethird strain was a species of Bacillus subtilis. About 80% of the sporeblend formulation was Bacillus licheniformis (40% of each strain) sporesand 20% of the spores in the spore blend formulation were Bacillussubtilis. The spore composition also included water, thickener, andorganic salts, according to a preferred embodiment described above.

The combined working solution (containing nutrient germinantcomposition, spore composition, and water) was incubated at about 42° C.for about 1 hour to produce an activated bacteria solution. Followingthis incubation, the entire activated bacteria solution (about 1 mL) wasadded to aquarium 3. Mixing was accomplished via aeration by mixingstone. Table 5 shows the composition of the starting nutrient germinantformulation. Table 6 shows the composition of the working solution thatwas incubated. After mixing the incubated bacteria solution into 55gallons of aquarium 3, the concentration of the bacteria was about9.6×10² CFU/mL and the final percent of the nutrient germinantcomposition in aquarium 3 was about 9.6×10⁻⁶% v/v. The contents of eachaquarium after their respective treatments have been applied are shownin Table 7. The trial continued for 120 days.

TABLE 5 Components of a Nutrient Formulation. Wt % in Starting g/L inStarting g/L in Working Nutrient Nutrient Nutrient-Spore g/L in FinalComponent Formulation Formulation formulation Dilution Water 82.9 829996.58 999.97 L-alanine 8.9 89 1.78  8.5 × 10⁻⁵ Disodium 6 60 1.2 5.76 ×10⁻⁶ Phosphate Monosodium 2 20 0.4 1.93 × 10⁻⁶ phosphate Germaben II 0.22 0.04 1.92 × 10⁻⁷

TABLE 6 Working Nutrient-Spore Formulation. Starting Final ComponentVolume Concentration Concentration Starting Nutrient  20 μL 100% (v/v) 2% (v/v) Formulation Starting Spore  20 μL 100% (v/v) about About 2 ×10⁸ Formulation 10¹⁰ CFU/ML CFU/mL Water  960 μL 100% (v/v) 96% (v/v)Final Volume 1000 μL NA NA

TABLE 7 Aquarium Contents (Treatment Groups) Aquarium 3 (Nutrient-Aquarium 1 Aquarium 2 (Pond Spore Formulation and (Control Powder OnlyPond Powder Treatment Group) Treatment Group) Treatment Group) Noadditions 0.5 g of Calcium 1 mL of activated bacteria Carbonate addedsolution (incubated per every water Working-Nutrient Spore replacementFormulation) and 0.5 g of Calcium Carbonate added per every waterreplacement

Table 8 shows the final weight and body measurements of the averagedtrial groups as well as standard deviations. The control group inaquarium 1 had the smallest shrimp weight and body measurements comparedto treatment groups in aquariums 2 and 3. Aquarium 3 had the largestshrimp and had the best results in terms of shrimp size compared to theprawns in aquariums 2 and 1. The average final weight of shrimp inaquarium 3 was 6.48 g. The average final weight of the prawns inaquarium 2 was 4.87 g on average. The average final weight of the prawnsin aquarium 1 (the control) was 3.43 g. The average total length forprawns in aquarium 3 was also the greatest at 7.98 cm. The average totallength for prawns in aquarium 2 was 7.41. The average total length forprawns in aquarium 1 (the control group) was 6.95. The average taillength of prawns in aquarium 3 was 4.67 cm. The average tail length ofprawns in aquarium 2 was 4.26 cm. The average tail length of prawns inaquarium 1 (control) was the smallest at 3.87 cm.

TABLE 8 Effect of Treatment on Growth Performance. Calcium Spores +Calcium Control Carbonate Carbonate Average final weight 3.43 4.87 6.48(g) Average total length 6.95 7.41 7.98 (cm) Average Tail length 3.874.26 4.67 (cm) Standard Deviation 0.77 4.11 4.76 Average Final WeightStandard Deviation 0.42 1.49 1.30 Average Total length StandardDeviation 0.27 0.68 0.81 Average Tail length

FIG. 10 shows an image of the three aquariums that can demonstrate thewater clarity in each group by the end of the 120 day trial. In terms ofwater clarity, aquarium 3 was observed to be clearest. Aquarium 1, thecontrol, was observed to have the greatest amount of algal growthcovering the aquarium walls as compared to aquariums 2 and 3. Aquarium 2was observed to have only moderate algal growth on the aquarium walls ascompared to the control and aquarium 3.

During the 120-day trial, all three aquariums started off with little tono algae on the sides of the aquariums. As the trial progressed, thecontrol aquarium (aquarium 1) accumulated more algal growth on the sidesof the aquarium (see e.g. FIG. 10). Aquarium 2 had less algal growththan aquarium 1. Aquarium 3 had little to no algal growth as compared toaquariums 1 and 2.

Water parameters were consistent throughout the trial. Ammonia levelswere zero for all three aquariums. Nitrite/nitrate were also within saferanges for the duration of the trial. pH also stayed within normalranges of about 7.5 to 8.5 for all of the aquariums. There were no waterparameter spikes observed that could have harmed the prawns as aquariumcycling occurred safely and parameters remained consistent for the fulllength of the 120-day trial.

Those of ordinary skill in the art will also appreciate upon readingthis specification and the description of preferred embodiments hereinthat modifications and alterations to the methods and nutrient germinantand spore compositions may be made within the scope of the invention andit is intended that the scope of the invention disclosed herein belimited only by the broadest interpretation of the appended claims towhich the inventors are legally entitled.

What is claimed is:
 1. A method of adding bacteria to water used in anaquaculture application, the method comprising: providing a volume ofnutrient germinant composition and a volume of bacteria spores, whichmay be premixed together as a nutrient spore composition or separate;optionally mixing a portion of the nutrient germinant composition and aportion of the bacteria spores if separate to form the nutrient sporecomposition; heating a portion of the nutrient spore composition to atemperature in a range of around 38° C. to 60° C. at or near a site ofthe aquaculture application; maintaining the temperature in the rangefor an incubation period of around 2 minutes to around 6 hours to form abatch of germinated bacteria solution; periodically repeating theheating and maintaining steps to form additional batches of germinatedbacteria solution over the course of a treatment cycle; dispersing eachbatch of germinated bacteria solution into the water used in theaquaculture application.
 2. The method of claim 1 wherein the bacteriaare one or more species of Bacillus licheniformis and Bacillus subtilis.3. The method of claim 1 wherein the bacteria is selected from the groupconsisting of Bacillus amylophilus, Bacillus licheniformis, Bacilluspumilus, Bacillus subtilis, Bacteriodes ruminocola, Bacteriodesruminocola, Bacterioides suis, Bifidobacterium adolescentis,Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacteriuminfantis, Bifidobacterium longum, Bifidobacterium thermophilum,Enterococcus cremoris, Enterococcus diacetylactis, Enterococcus faecium,Enterococcus intermedius, Enterococcus lactis, Enterococcusthermophiles, Lactobacillus brevis, Lactobacillus buchneri,Lactobacillus bulgaricus, Lactobacillus casei, Lactobacilluscellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii,Lactobacillus farciminis, Lactobacillus fermentum, Lactobacillushelveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillusreuteri, Leuconostoc mesenteroides, Megasphaera elsdennii, Pediococcusacidilacticii, Pediococcus cerevisiae, Pediococcus pentosaceus,Propionibacterium acidipropionici, Propionibacterium freudenreichii, andPropionibacterium shermanii.
 4. The method of claim 1 wherein thenutrient germinant composition comprises: an L-amino acid; one or morebuffers comprising a phosphate buffer, HEPES, Tris base, or acombination thereof; an industrial preservative; optionally D-glucose,or optionally D-fructose, or optionally both D-glucose and D-fructose;and optionally a source of potassium ions.
 5. The method of claim 4wherein the L-amino acid is L-alanine, L-asparagine, L-valine,L-cysteine, a hydrolysate of soy protein, or a combination thereof andwherein the nutrient germinant composition comprises around 17.8g/L to89 g/L total of one or more L-amino acids.
 6. The method of claim 5wherein the nutrient germinant composition and spores are premixed andthe nutrient spore composition further comprises spores of a Bacillusspecies and a germination inhibitor.
 7. The method of claim 6 whereinthe germination inhibitor or preservative comprises sodium chloride,D-alanine, or a combination thereof.
 8. The method of claim 7 whereinthe premixed nutrient spore composition comprises around 29 g/L to 117g/L sodium chloride and/or.around 8 g/L to 116 g/L D-alanine.
 9. Themethod of claim 5 wherein the phosphate buffer comprises around 10-36g/L of monosodium phosphate and around 30-90 g/L of disodium phosphate10. The method of claim 1 wherein the nutrient germinant composition, ornutrient spore composition if premixed, is a concentrated liquidcomprising: around 8.9-133.5 g/L of one or more L-amino acids; around0.8-3.3 g/L total of one or more industrial preservatives; around 40-126g/L total of one or more phosphate buffers, around 15-61 g/L Tris base,or around 32.5-97.5 g/L HEPES, or a combination thereof; optionallyaround 18-54 g/L of D-glucose, D-fructose, or a combination thereof; andoptionally around 7.4-22.2 g/L of KCl.
 11. The method of claim 10further comprising: adding a diluent to the nutrient germinantcomposition, or nutrient spore composition if premixed, prior to orduring heating; and mixing the diluted nutrient-germinant compositionand bacteria spores, or diluted nutrient spore composition, during theincubation period; and wherein the concentration of the dilutednutrient-germinant composition is around 0.1% to 10%.
 12. The method ofclaim 1 wherein the bacteria spores are in a separate spore compositioncomprising: one or more Bacillus species in spore form; about 0.002 to5.0% by weight thickener; about 0.01 to 2.0% by weight total of one ormore acids or salts of acids; and wherein the percentages are by weightof the spore composition.
 13. The method of claim 4 wherein the bacteriaspores are in a separate spore composition comprising: one or moreBacillus species in spore form; one or more acids or salts of acids; athickener; and wherein the spore composition has a pH of around 4.5 toaround 5.5.
 14. The method of claim 13 wherein the spore Bacillusspecies are one or more of wherein the bacterial species comprises oneor more of: Bacillus pumilus, Bacillus licheniformis, Bacillusamylophilus, Bacillus subtilis, Bacillus amyloliquefaciens, Bacillusclausii, Bacillus firmus, Bacillus megaterium, Bacillus mesentericus,Bacillus subtilis var. natto, or Bacillus toyonensis.
 15. The method ofclaim 13 wherein the acids or salts of acids are one or more of aceticacid, citric acid, fumaric acid, propionic acid, sodium propionate,calcium propionate, formic acid, sodium formate, benzoic acid, sodiumbenzoate, sorbic acid, potassium sorbate, or calcium sorbate.
 16. Themethod of claim 13 wherein the spore composition comprises: about 0.002to 5.0% by weight thickener; about 0.01 to 1% by weight total of one ormore acids or salts of acids; and about 0.00005 to 3.0% by weight of asurfactant; wherein the percentages are by weight of the sporecomposition.
 17. The method of claim 3 wherein the incubation period isaround 2 minutes to around 5 minutes.
 18. The method of claim 17 whereinthe aquaculture application is a growing pond containing fish or eel.19. The method of claim 1 wherein the incubation period is around 4 to 6hours.
 20. The method of claim 19 wherein the aquaculture application isa growing pond containing shrimp.
 21. The method of claim 1 wherein thetemperature is in a range of around 38° C. to 50° C.
 22. The method ofclaim 1 wherein the temperature is in a range of around 41° C. to 44° C.23. The method of claim 1 wherein the nutrient germinant compositiondoes not include fructose or glucose.
 24. The method of claim 4 whereinthe industrial preservative comprises methyl chloro isothiazolinone,methyl isothiazolinone, propylparaben, methylparaben, diazolidinyl urea,or a combination thereof.
 25. The method of claim 1 wherein the bacteriacomprise two strains of Bacillus licheniformis and one strain ofBacillus subtilis.